Compositions and methods for treating malignancies

ABSTRACT

Provided by the invention are methods for identifying therapeutic agents for treating multiple myeloma or another hematological malignancy, as well as methods for determining the prognosis of a patient with multiple myeloma or another hematological malignancy. The methods are based in part on the inventors&#39; discovery that an extracellular form of cyclophilin A binds to CD147 expressed on multiple myeloma cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. § 371 ofPCT/US15/53460 filed on Oct. 1, 2015, which claims priority to U.S.Provisional Application No. 62/058,911 filed on Oct. 2, 2014 and U.S.Provisional Application No. 62/119,377 filed on Feb. 23, 2015. Thecontents of each of the aforementioned patent applications are herebyincorporated by reference in their entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant 1186004awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

The invention relates generally to compositions and methods for treatingmalignancies, and more particularly to compositions and methods fortreating a hematological malignancy such as multiple myeloma.

SUMMARY

The invention is based in part on the discovery that the extracellularform of cyclophilin A (eCyPA) is secreted at high levels by bone marrowendothelial cells. In addition, eCyPA promotes migration andproliferation of multiple myeloma (MM) cells and effects homing of MMcells to the bone marrow by binding to the CD147 receptor on MM cells.

In one aspect, the invention relates to a method for identifying anagent for treating multiple myeloma (MM). The method comprises providinga first polypeptide comprising a CD147 polypeptide and a secondpolypeptide comprising an extracellular cyclophilin A (eCyPA)polypeptide sequence under conditions that allow for binding of theCD147 polypeptide and the eCyPA sequence. The complex is then contactedwith a test agent. The method further includes determining whether thetest agent disrupts binding of the first and second polypeptide.Disruption of binding of the first polypeptide and second polypeptide bythe test agent indicates the test agent is a therapeutic agent fortreating MM.

In another aspect, the invention relates to a method for identifying anagent for treating chronic lymphocytic leukemia (CLL) andlymphoplasmacytic lymphoma (LPL), the method comprising

providing a first polypeptide comprising a CD147 polypeptide and asecond polypeptide comprising an extracellular cyclophilin A (eCyPA)polypeptide sequence under conditions that allow for binding of theCD147 polypeptide and the eCyPA sequence;

contacting the complex with a test agent; and

determining whether the test agent disrupts binding of the first andsecond polypeptide, where the disruption of the binding of the firstpolypeptide and second polypeptide by the test agent indicates the testagent is a therapeutic agent for treating MM.

In a still further aspect, the invention relates to a method ofdetermining a prognosis for a subject with multiple myeloma (MM), themethod comprising;

providing a sample from a subject with MM;

assaying the sample to determine a level of eCyPA in the sample toobtain an eCyPA test value; and

comparing the eCyPA test value to an eCyPA reference value calculatedfor a sample whose MM status is known, where an eCyPA test value greaterthan a reference eCyPA value in a sample known not to have MM indicatesthat the subject has a poor prognosis, and where an eCyPA test valueless than a reference eCyPA value in a sample known to have MM indicatesthat the subject has a good prognosis.

In yet another aspect, the invention relates to a method for determiningefficacy of a multiple myeloma treatment in subject, the methodcomprising

providing a sample from a subject with MM;

assaying the sample to determine a level of eCyPA in the sample toobtain an eCyPA test value; and

comparing the eCyPA test value to an eCyPA reference value calculatedfor a sample whose MM status is known; wherein

an eCyPA test value greater than a reference eCyPA value in a referencesample known not to have MM indicates that the treatment is notefficacious, and

an eCyPA test value less than a reference eCyPA value in a sample knownto have MM indicates that the treatment is efficacious.

In another aspect, the invention relates to a method of diagnosingmultiple myeloma (MM) or a multiple myeloma precursor condition in asubject, the method comprising

providing a sample from a subject;

assaying the sample to determine a level of eCyPA in the sample toobtain an eCyPA test value; and

comparing the eCyPA test value to an eCyPA reference value calculatedfor a sample whose MM status is known, wherein

an eCyPA test value greater than a reference eCyPA value in a referencesample known not to have MM indicates that the subject has MM or a MMprecursor conditions, and

an eCyPA test value equal to or less than a reference eCyPA value in asample known to have MM indicates that the subject does not havemultiple myeloma.

In a further aspect, the invention relates to a method of determiningthe progression of multiple myeloma (MM) in a subject, the methodcomprising;

providing a sample from a subject known to or suspected of having amultiple myeloma;

assaying the sample to determine a level of eCyPA in the sample toobtain an eCyPA test value; and

comparing the eCyPA test value to an eCyPA reference value calculatedfor a sample whose blood cancer stage is known,

wherein an eCyPA test value greater than a reference eCyPA value in asample known not to have MM indicates that the subject has a MM moreadvanced than MM in subjects from which the reference eCyPA value iscalculated, and

an eCyPA test value less than the reference eCyPA value indicates thatthe subject has a MM less advanced than MM in subjects from which thereference eCyPA value is calculated.

In another aspect, the invention relates to a method of preparing atherapeutic agent for treating multiple myeloma (MM) in a subject themethod comprising:

providing a DNA encoding the variable domains of a donor CyPA antibody;

determining the amino acid sequence of the CDR regions of the donormonoclonal antibody from the DNA;

selecting human acceptor antibody sequences; and

producing a humanized CyPA antibody comprising the CDRs from the donorantibody and variable region frameworks from the human acceptor antibodysequences.

In yet another aspect, the invention relates to a composition comprisinga biocompatible substrate and bone marrow-derived endothelial cells(BMEC) admixed with a biocompatible substrate, where the BMEC cellsexpress eCyPA in an amount sufficient to stimulate proliferation ormigration of multiple myeloma (MM) cells.

The methods described herein can also be used to determine tumor burdenin a patient with multiple myeloma. The inventors have discovered thatCyPA serum levels are not only associated with MM progression, such asincreasing level from MGUS, to SMM, to MM, but are also associated withtumor burden, such as decreasing level from MM to progressed (treatedpatients). Accordingly, CyPA can be used to monitor MM burden andprogression in different stages of multiple myeloma progression, as wellfor indicating the amount of tumor burden remaining after treatment.

The invention additionally provides methods for identifying inhibitorsof viral infections by inhibiting or otherwise reducing levels ofintracellular and/or extracellular CyPA. Agents that reduceintracellular levels of CyPa can also be tested for their ability toreduce extracellular levels of CyPA. Similarly, agents that reduceextracellular levels of CyPa can also be tested for their ability toreduce intracellular levels of CyPA.

In another aspect, the invention relates to a method for identifying anagent for inhibiting viral infections. The method includes

providing a cyclophilin A (CyPA) polypeptide sequence;

contacting the complex with a test agent; and

determining whether the test agent binds to the CyPA polypeptidesequence, wherein binding of the test agent to CyPA indicates the testagent is an inhibitor of viral infection.

In some embodiments, the CyPA is provided external to a cell, forexample, as a secreted polypeptide from a cell expressing CyPA. In otherembodiments, the CyPA is provided in a cell.

In some embodiments, the method further includes determining whetherbinding of the test agent to CyPA lowers CyPA, e.g., in a cell freebodily fluid such as serum, where it may be present as an extracellularsecreted polypeptide.

The invention additionally provides methods for identifying inhibitorsof viral infections by inhibiting or otherwise reducing levels ofintracellular and/or extracellular CyPA. The method includes providing acyclophilin A (CyPA) polypeptide sequence; contacting the complex with atest agent; and determining whether the test agent blocks interactionsbetween the CyPA polypeptide sequence and a CD147 polypeptide. Blockingor otherwise disrupting the interaction between CyPA and the CD147polypeptide indicates the test agent is an inhibitor of viral infectionor a therapeutic agent for treating MM. The CD147 polypeptide can beprovided on the surface of a cell or in a cell-free solution orsubstrate.

If desired, the test agent can be screened for its ability to loweramounts of or otherwise inhibit both intracellular and extracellularCyPA polypeptide levels. Among the advantages of the invention is thatit provides convenient and relatively non-invasive tests for diagnosing,staging, or determining the prognosis of, or otherwise assessingmultiple myeloma in a patient. The assays can be performed using aperipheral blood sample without the need to make an incision.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention are apparent from thefollowing description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Some of the figures in the application were executed in color.

FIG. 1. Analysis of BCL9 expression and canonical Wnt activity in BMECs.(A) Representative CD34 immunostains in BM biopsies from normalindividuals (NBM) (n=20) as well as MGUS (n=20) and MM patients (MMPT)(n=60). (B) Representative immunohistochemical analysis of BCL9expression (brown color) in endothelial cells (arrows) in BM biopsiesfrom MM patients (MMPT) or normal bone marrow (NBM) from otherwisehealthy subjects. Selected representative cases are shown. Anti-CD138staining (red color) is used as a marker of plasma cells on the leftpanel (arrows). Anti-CD34 staining (red color) is used as a marker ofendothelial cells (right bottom panel). Immunoblots (C) andimmunofluorescence (D) analysis of BCL9 (left panel) and β-cateninexpression (middle panel) in primary endothelial cells derived from BMfrom two MM patients (PBMEC#1, PBMEC#1) and two BM endothelial celllines (BMEC-1, BMEC-60). Note co-expression of BCL9 and β-catenin byimmunoblotting and by nuclear co-localization immunofluorescence (rightpanel). Factor VIII is used as marker of endothelial cells inimmunoblots. (E) Wnt reporter activity of BMEC-1, BMEC-60 and PBMEC #1cells lentivirally transduced with shRNAs against BCL9 (BCL9-shRNA)compared with cells lentivirally transduced with scrambled shRNAs(Control). (F) Proliferation of BMEC-1, BMEC-60 and PBMEC #1 cellstreated with medium alone (Vehicle) or in the presence of 10 uMStabilized Alpha Helix peptides of BCL9 SAH-BCL9 (P<0.006).Proliferation and Wnt reporter data was normalized based on control orvehicle data. Results are means±SD for assays performed in triplicate.Statistical significance of differences between groups was determined byapplying the unpaired Student's t-test.

FIG. 2. Biochemical and functional analysis of MM cells upon interactionwith BMECs. Immunoblot analysis of total protein extracts from H929 andMM1S cells incubated in the absence (−) or presence (+) of BMEC-60 cellsin the same (A) or separate (B) chambers (transwells). (C) Immunoblotanalysis of total protein extracts from MM1S cells incubated in theabsence (−) or presence (+) of endothelial cells derived from BM fromtwo different MM patients (PBMEC #1, PBMEC #2) using transwell chambers.Cell proliferation assays of MM1S cells (D) and MM cells from twodifferent MM patients (E, MMPT #1 MMPT #2), and incubated in the absence(−) or presence (+) of BMEC-60 cells using transwell chambers. (F) Cellviability assays using the tumor cell-specific in vitro bioluminescenceimaging (CS-BLI) 44 of MM1S-luc cells incubated in the presence orabsence of increasing concentrations of doxorubicin (top) ordexamethasone (bottom), without (−) or with (+) BMEC-60 cells. (G)Proliferation of MM1S and H929 cells in the absence (−) or presence (+)of endothelial cells from BM of two different MM patients (PBMEC #1,PBMEC #2). (H) Immunoblot analysis of total protein extracts from H929and MM1S cells incubated in transwell chambers in the presence ofγ-irradiated BMEC-60 cells lentivirally transduced with either scrambledshRNAs (Control) or shRNAs against BCL9 (BCL9-shRNAs). (I) Knockdownexpression of BCL9 in γ-irradiated BMEC-60 cells was associated withreduced proliferation of co-cultured MM1S cells. Proliferation data wasnormalized based on control data. Results are means±SD for assaysperformed in triplicate. Statistical significance of differences betweengroups was determined by unpaired Student's t-test.

FIG. 3. In vitro and in vivo migration of MM cells toward BMECs.Transwell migration assays of MM1S-luc cells incubated under differentconditions: (A) growth in medium alone (medium), conditioned medium fromBMEC-60 cells (BMEC-60-CM) or conditioned medium derived from BMEC-60cells and treated with proteinase K (BMEC-60 CM+PK); (B) growth in theabsence or presence of endothelial cells derived from BM from twodifferent MM patients (PBMEC #1, PBMEC #2); (C) growth in the presenceof HS5 cells or PBMEC #1 and PBMSC #1 isolated from same patient.Migration data was normalized based on data of medium alone. Results aremeans±SD for assays performed in triplicate. Statistical significance ofdifferences between groups was determined by unpaired Student's t-test.(D) Diagram of the three-dimensional poly-ε-caprolactone scaffoldxenograft mouse model. Xenogen data (E), time course (F), and histologicanalysis (G) of MM1S-luc cell growth within non-coated scaffolds(orange) or within scaffolds coated with HS5 (green) or BMEC-60 (blue)cells. ERG (Ets-related gene): Endothelial cell marker. Xenogen data(H), time course (I), and histologic analysis (J) of MM1S-luc cellgrowth within scaffolds coated with primary BM endothelial cells (PBMEC#1 and PBMEC #2) or primary BM stromal cells (PBMSC #1 and PBMEC #2)isolated from same MM patient. Statistical analysis of tumor burden wasdone using factorial analysis in SPSS 13.0. The results of tworepresentative experiments of three are shown

FIG. 4. Secretion of eCyPA and eCyPB by BMEC and increased BM serumlevels of eCyPA in MM patients. (A) Transwell migration assays ofMM1S-luc cells incubated in the presence of HS5 cells or BMEC-60 cellslentivirally transduced with scrambled shRNAs (Control-shRNA) or shRNAsagainst BCL9 (BCL9-shRNA). Xenogen data (B), time course (C), andhistologic analysis (D) of MM1S-luc cell growth within scaffolds coatedwith BMEC-60 cells lentivirally transduced with scrambled shRNAs(Control-shRNA) or shRNAs against BCL9 (BCL9-shRNA). (E) Histogramrepresentation of proteins identified by mass spectrometric analysis ofexcised bands (blue) and whole protein supernatants from BMEC-60transduced with Control-shRNA (pink), as well as PBMEC #1 (yellow) andPBMEC #2 (green) cells. At the intersection of the diagram is eCyPA andeCyPB identified by both procedures. (F) ELISA of eCyPA and eCyPB levelsin CM from HS5 and BMEC-60 cells lentivirally transduced withControl-shRNAs or BCL9-shRNA. CM was taken after 24 hrs incubation inPBS. (G) ELISA of eCyPA and eCyPB in CM from primary BM endothelialcells (PBMEC #1 and PBMEC #2) or primary BM stromal cells (PBMSC #1 andPBMSC #2) isolated from same MM patient. Results are means±SD for assaysperformed in triplicate. Statistical significance of differences betweengroups was determined by unpaired Student's t-test. CM was taken after72 hrs culture. (H) Representative immunohistochemical stains of CyPAand CyPB expression in BM from healthy subjects (NBM) (n=20) and MMpatients (n=60) (MMPT). Black and yellow arrows indicate expression ofCyPA or CyPB in BMECS and myeloid cells, respectively, in a NBM. ELISAquantification of eCyPA (I) and eCyPB (J) levels in serum from BM and PBisolated from same MM patients (n=12).

FIG. 5. eCyPA promotes signaling changes, migration, and proliferationof MM cells. Transwell migration assays of MM1S-luc cells incubatedunder different conditions: (A) increased concentrations of recombinanteCyPA; (B) medium alone or BMEC-60 cells lentivirally transduced withControl-shRNA or shRNAs against CyPA (CyPA-shRNA). Migration data wasnormalized based on data of medium alone. Results are means±SD forassays performed in triplicate. Statistical significance of differencesbetween groups was determined by unpaired Student's t-test. (C)Immunoblot of total protein extracts from H929 and MM1S cells incubatedin the absence (−) or presence (+) of eCyPA at 50 ng/ml. (D) Immunoblotof total protein extracts from MM1S cells treated with increasingconcentrations of recombinant eCyPA (top) or 50 ng/ml of recombinanteCyPA for different times (bottom). (E) Immunoblot of total proteinextracts from H929 and MM1S cells incubated with BMEC-60 cellslentivirally transduced with Control-shRNA or CyPA-shRNA. (F)Proliferation analysis of H929 and MM1S cells incubated in the absenceor presence of increasing concentrations of eCyPA. Xenogen data (G),time course (H), and histologic analysis (I) of MM1S-luc cell growthwithin scaffolds coated with BMEC-60 lentivirally transduced withControl shRNAs or CyPA-shRNA. Statistical analyses of tumor burden weredone using factorial analysis in SPSS 13.0. The results of onerepresentative of three independent experiments is shown.

FIG. 6. CyPA promotes migration and growth of MM through the CD147receptor. (A) Transwell migration assays of MM1S-luc cells lentivirallytransduced with control-shRNAs or shRNAs against CD147 (CD147-shRNA) andincubated with medium alone or BMEC-60 cells or recombinant eCyPArecombinant proteins. (B) Immunoblot of total protein extracts from H929and MM1S cells lentivirally transduced with Control shRNA or CD147-shRNAin the presence of 50 ng/ml of recombinant eCyPA. Xenogen data (C), timecourse (D), and histologic analysis (E) of cell growth of MM1S-luctransduced with Control-shRNA or CD147-shRNA within empty scaffolds orscaffolds coated with BMEC-60 cells. (F) Transwell migration assays ofMM1S-luc cells incubated with medium containing none or 50 ng/ml ofeCyPA and increasing concentrations of CD147 Ab. (G) Transwell migrationof MM1S-luc cells incubated in the presence of medium alone, or PMMEC #1or PBMSC #1 with CD147 Ab (100 ug/ml) or CXCR4 Ab (100 ug/ml). Migrationdata was normalized to medium alone. Results are means±SD for assaysperformed in triplicate. Statistical significance of differences betweengroups was determined by unpaired Student's t-test.

FIG. 7. Targeting eCyPA/CD147 complex is associated with anti-MMactivity. Decreased CD147 expression in circulating MM cells. Xenogendata (A) and histologic analysis (B) of MM1S-luc cell growth withinscaffolds coated with BMEC-60 cells and implanted subcutaneously in CB17.Cg-PrkdcscidLystbg-J/Crl mice. Groups of 4 mice were subsequentlytreated with either isotype Ab or anti-CD147 Ab, and tumor growth withinthe scaffolds was evaluated by Xenogen imaging every five days. (C)Immunofluorescence analysis of CD147 expression in MM plasma cells fromBM (top) and peripheral blood (PB) (bottom) from one MM patient (Case#1). (D) Immunofluorescence analysis of CD147 expression in normalplasma cells from BM (top) and lymph node (LN) (bottom) in two differentnormal donors (Case #3 and #4).

FIG. 8. Proposed model of BM homing of MM cells based on eCyPA secretedby BMECs and on CD147 expression by MM cells.

FIG. 9. (A) Representative BCL9 immunostains (brown) in BMECs fromnormal individuals (NBM) (n=20) and MM patients (n=60). (B) BCL9immunoblots in total protein extracts from BMEC-60 and BMEC-1 cellslentivirally transduced with control-shRNA or BCL9-shRNAs. Actin wasused as a loading control. (C) Proliferation of BMEC-60 cellslentivirally transduced with Control-shRNA or BCL9-shRNA. (D) Immunoblotanalysis of Factor VIII and CyPA expression in primary BMECs (PBMEC #1and PBMEC #2) and primary BMSCs (PBMSC #1 and PBMSC #2) isolated fromthe same MM patient. (E) Transwell migration assays of U266 and RPMIcells incubated in the presence of medium alone or HS5, PBMSC #1 orPBMEC #1 cells. (F) Left and right, results of two independentexperiments shown in FIGS. 3E-G. Xenogen data (G), time course (H), andhistologic analysis (I) of H929-luc cell growth within scaffolds coatedwith PBMEC #1 cells, but not in uncoated, scaffolds.

FIG. 10. (A) Histologic analysis of primary MM cell growth (MMPT #1 andMMPT #2) in scaffolds coated with BMEC-60 cells, but not in uncoatedscaffolds in the scaffold mouse xenograft model. (B) Transwell migrationof H929 cells co-cultured with BMEC-1 cells lentivirally transduced withControl-shRNA or BCL9-shRNAs. (C) and (D) display data from twoexperiments shown in FIGS. 4B-D. Xenogen data (E), time course (F), andhistologic analysis (G) of H929-luc cell growth within scaffolds coatedwith BMEC-60 cells lentivirally transduced with Control-shRNA orBCL9-shRNA #2. (H) Silver-stained agarose gels of proteins secreted byBMEC-60 cells lentivirally transduced with Control-shRNA or BCL9-shRNAs,PBMEC #1, PBMSC #1 or HS5 cells. Bars indicate low-molecular weightbands present in CM from BMEC-60 cells transduced with control-shRNA butnot transduced with BCL9-shRNA, which were excised and analyzed by massspectrometry. (I) Immunoblot analysis of BCL9, CyPA and CyPB expressionin total protein extracts from BMEC-60 cells transduced withcontrol-shRNA or BMEC-60 cells transduced with BCL9-shRNAs.

FIG. 11. (A) Representative CyPA (top) and CyPB (bottom) immunostains innormal bone marrows (n=20). (B) Double immunofluorescence analysis ofCyPA (green) and myeloperoxidase (red) expression in BM aspirate fromnormal individual. (C) Representative CyPA (top) and CyPB (bottom)immunostains in BM from MM patients (n=60). (D) Time course ELISAanalysis of CyPA secretion by the indicated cells. 5×10⁵ cell wereplated in each case. Results are means±SD for assays performed intriplicate.

FIG. 12. (A) CyPA immunostains in two different NBM, kidney, liver, andlymph node biopsies from healthy subjects. (B) Serum levels of eCyPA in10 BM aspirates from MGUS and MM patients. (C) Transwell migration assayof MM1S-luc cells incubated in the absence of serum (Medium) or BM serumfrom two different MM patients with low (PT#4) and high (PT#12) levelsof eCyPA (same as FIG. 4I). Migration data was normalized based on dataof medium alone. (D) Transwell migration assay of H929, OPM2 and RPMIcells incubated with medium alone or in the presence of 50 ng/ml ofrecombinant eCyPA. (E) Transwell migration assay of two MM primarytumors (MMPT #1 and MMPT #2) incubated with medium alone or 50 ng/ml ofrecombinant eCyPA or BMEC-60 cells.

FIG. 13. (A) Transwell migration assay of MM1S-luc cells incubated withmedium alone or 50 ng/ml of recombinant eCyPA or eCyPB. (B) Immunoblotof CyPA and CyPA expression in BMEC-60 and BMEC-1 cells lentivirallytransduced with control-shRNAs or CyPA-shRNAs. (C) Transwell migrationassay of MM1S cells co-cultured with BMEC-1 cells lentivirallytransduced with control-shRNA or CyPA-shRNAs. (D) Transwell migrationassay of MM cells co-cultured with BMEC-1 cells lentivirally transducedwith control-shRNA or CyPA-shRNAs, in the absence or presence of 50ng/ml of recombinant eCyPA. (E) Top and bottom, two independentexperiments with results shown in FIGS. 5G-I. Xenogen (F), time course(G) and histological analysis (H) of H929-luc cell growth in scaffoldscoated with BMEC-60 cells lentivirally transduced with control-shRNA orCyPA-shRNA #1.

FIG. 14. (A) Flow cytometry of CD147 (red line) and control isotype(black line) expression in six different MM cell lines. (B) Top: CD147immunostain in three representative cases of BM biopsies from MMpatients (n=60). Bottom: flow cytomety of CD147 in MM cells from threerepresentative BM aspirates from MM patients (n=10). (C) mRNA expressionof CD147 (GSE6477) in BM plasma cells from a normal subjects (12) and MMpatients (n=60). (D) Immunoblots of CD147 in total protein extracts fromH929 and MM1S cells lentivirally transduced with control-shRNA orCD147-shRNA. (E) Transwell migration assay of H929 and MM1S cellco-cultured with BMEC-60 cells lentivirally transduced withcontrol-shRNA or CD147-shRNA. (F) Immunoblot analysis of pSTAT, pAKT,pERK and PARP in MM1S cells incubated with medium alone, 50 ng/ml ofrecombinant eCyPA, 100 ug/ml of CD147 Ab, or 50 ng/ml of recombinanteCyPA plus 100 ug/ml of CD147 Ab. (G) Immunoblot analysis of totalprotein extracts from MM1S cells incubated with two differentconcentrations of CD147 Ab. (H) Transell migration assay of MM1S-luccells incubated with medium alone, or 50 ng/ml of recombinant eCyPA, ora combination of either 50 ng/ml of recombinant CyPA plus 100 ug/ml ofCD147 Ab or 50 ng/ml or recombinant CyPA plus 100 ug/ml of CXCR4 Ab. (I)ELISA assay of CyPA and SDF-1 in CM from the indicated primary cells.5×10⁵ cell were plated for each cell and incubated for 72 hrs. Resultsare means±SD for assays performed in triplicate.

FIG. 15. (A) Time course of Xenogen imaging of MM1S-luc cell growth inscaffolds implanted in CB17.Cg-PrkdcscidLystbg-J/Crl mice and treatedwith local injections of isotype control or anti-CD147 Abs. (B)Complement dependent cytotoxicity (CDC) was assessed usingCalcein-AM-labeled MM1S-Luc cells, CD147 Ab, isotype control Ab, andmouse serum. (C) Immunofluorescence analysis of CD147 expression in MMplasma cells from bone marrow (BM) (top) and peripheral blood (PB)(bottom) from a MM patient (Cases #2). (D) Immunofluorescence analysisof CD147 expression in normal plasma cells from bone marrow (BM) (top)and lymph node (LN) (bottom) from two different normal donors (Cases #5and #6). For each sample from BM, PB, and LN from MM patients or normalindividuals, 10 different fields (a-j) with plasma cells were analyzed.Only two representative fields are shown (a, b).

FIG. 16. (A) Representative immunostains (top, n=10) and flow cytometric(bottom, n=6) analysis of CD147 expression in BM samples from CLLpatients. (B) Representative immunostains (top, n=10) and flowcytometric (bottom, n=6) analysis of CD147 expression in BM samples fromLPL patients. (C) Flow cytometry of CD147 (red line) and control isotype(black line) expression in one CLL cell line (HG3) and one primary tumorpatient cells (CLLPT #1). (D) Flow cytometry of CD147 (red line) andcontrol isotype (black line) expression in one LPL cell line (BCWM.1)and one primary tumor patient cells (LPLPT #1). (E) Transwell migrationassay of HG3 (left) and CLLPT#1 (right) cells incubated with mediumalone, BMEC-60 cells, or 50 ng/ml of recombinant eCyPA. (F) Transwellmigration of BCWM.1 (left) and LPLPT #1 (right) cells incubated withmedium alone, BMEC-60 cells, or 50 ng/ml of recombinant eCyPA. Migrationof primary CLL and LPL tumor patient cells was evaluated in fourdifferent samples for each tumor type. One representative sample foreach primary tumor type is shown. Unlabeled cell lines and primarytumors were used, and migration was evaluated by counting cells in thebottom chamber. Representative fields are shown at the bottom.

FIG. 17 is a graphical representation of an ELISA assay showing eCyPA(ng/ml) in peripheral blood serum from four different patentgroups—MGUS, SMM, MM and MM treated (n=25 per group). eCyPA level iscorrelated with progress of multiple myeloma, from MGUS to MM stage andis decreased after treatment. eCyPA could use a biomarker to monitor MMprogress. MGUS: Monoclonal gammopathy of unknown significance; SMM:Smoldering multiple myeloma; MM: multiple myeloma; MM treated: patientunder treatment.

DETAILED DESCRIPTION OF THE INVENTION

Metastasis of epithelial tumors is a complex and poorly understoodmolecular, cellular, and organismal multistage process. Sometimes termedthe invasion-metastasis cascade, it occurs when cancer cells spread fromthe site of origin to anatomically distant organs. The process is drivenby acquisition of genetic and/or epigenetic alterations within the tumorcells themselves (the ‘seeds’), and by multistep interplay of thesecells with the host microenvironment (the ‘soil’), culminating incolonization in a foreign organ. Akin to certain epithelial neoplasms(e.g., prostate and breast carcinomas) that frequently metastasize andcolonize to the bone marrow (BM), B-cell lymphoid malignancies such asmultiple myeloma (MM), chronic lymphocytic lymphoma (CLL), andlymphoplasmacytic lymphoma (LPL), preferentially colonize and accumulatewithin the BM. Although the molecular mechanisms responsible for thispreferential colonization are still not completely understood,pathogenetic studies indicate that the BM-microenvironment (BM-ME),comprised of an extracellular matrix and diverse cellular elements (e.g.stromal, adiposal, endothelial, and hematopoietic), plays a pivotalrole. As with the BM homing of hematopoietic stem cell cells (HSCs),migration of MM and other B-cell malignancies from the PB to the BMniche is not a passive process, but rather is a complex active processrequiring multiple adhesion and chemokine receptors. BM homing of HSCsinvolves tethering of the cells by E- and P-selectins, associated withP-selectin glycoprotein ligand-1 and CD44 on the cells. This tetheringinvolves interaction of endothelial cells with circulating HSCs, andleads to rolling of the HSCs along the endothelium and activation of theSDF-1/CXCR4 axis, followed by VLA-/VCAM-1 activation. Other moleculesthat appear to play a role in HSCs homing include LFA-1, VLA-5, andactivated metalloproteases MMP2/9.

Here we have used MM as a prototypical terminally differentiated B-cellneoplasm to investigate the cellular and molecular BM-ME propertiesinvolved in BM colonization. MM is a fatal hematological malignancy ofterminally differentiated, post-germinal B-cells that originate in thelymph nodes and accumulate in the BM during disease evolution.Reciprocal interactions between MM cells and the BM-ME not only mediatetheir growth, but also protect them from apoptosis, resulting in thebone lytic lesions and BM angiogenesis. Interestingly, however, at theend-stage of disease MM cells are able to survive and proliferate evenin the absence of the BM-ME. During this stage the number of MM cellscirculating in the PB increases, and growth outside the BM can occur.Similarly to the HSC, factors implicated in BM homing of MM include theCXCR4/SDF-1 axis, IGF-1, and intracellular regulators downstream ofCXCR4, including Rho and Rac. Of these factors, the CXCR4/SDF-1 axisplays an especially critical role in regulating migration and adhesionof MM cells.

Among the interactions between MM cells and the BM-ME, intimate physicalcontact with BMECs is a major feature and is most readily discernedduring early disease stages, when the tumor burden is low.Clinico-pathologic correlations which highlight the importance of thefunctional interactions between MM cells and BMECs include thefollowing: (1) BM angiogenesis is associated with MM cell growth,disease progression, and patient survival; (2) progression of monoclonalgammopathy of undetermined significance (MGUS), to active MM isassociated with increasing angiogenesis; and (3) microvascular densitycorrelates with disease stage and is a prognostic factor in newlydiagnosed MM patients receiving conventional and high-dose chemotherapy.

Although several pro-angiogenic molecules secreted by MM cells have beenidentified (e.g. VEGF, βFGF, and HGF), the signaling molecules secretedby BMECs, which that promote MM disease progression and BM homing arenot fully known.

We have investigated the role of BMECs in the colonization of MM cellsto the BM niche. Having previously observed high BCL9 expression inBMECs, but not other BM cells, we chose to focus on the role of thistranscriptional co-activator of the canonical Wnt/β-catenin pathway. Weused an integrated approach combining in vitro assays with in vivomigration assays that simulate the human-human heterotypic interactionsbetween MM and BM cells. Additionally, we performed proteomic analysisof signaling molecules secreted by BMECs, as well as shRNA-basedloss-of-function assays, to identify and functionally validate eCyPA asa novel transcriptional target of the Wnt/β-catenin/BCL9 complex. eCyPAis secreted by BMECs and promotes pleiotropic signaling changes thatenhance not only migration of MM cells toward the BM, but alsoproliferation mediated by binding to CD147 receptors on the MM cells. Acomparison between BMECs and BMSCs from the same MM patient demonstratedthat these cells play different roles in the migration and BMcolonization of MM cells. In contrast to primary BMECs, primary BMSCssecrete very little eCyPA but instead secrete SDF-1, thereby promotingmigration and BM homing of MM cells, less efficiently than primaryBMECs. Consistent with this finding, BMEC-induced migration of MM cellswas inhibited by a CD147 Ab, but not by a CXCR4 Ab. In addition,inhibition of the eCyPA/CD17 axis supressed migration, tumor growth, andBM-colonization in a mouse xenograt model of MM. Furthermore, we havedocumented that eCyPA promotes migration of CLL and LPL cells, two otherB-cell malignancies that colonize the BM and express CD147. Takentogether, our findings indicate that cells within the BMmicroenvironment play different roles in MM progression, and offer apotential link between chronic inflammation, immunomodulation, and thepathogenesis of MM, CLL and LPL. Moreover, our results provide acompelling rationale for exploring the role of eCyPA and CD147 asmarkers of disease progression and for the development of noveltherapeutic approaches based on targeting the eCyPA/CD147 signalingcomplex.

Screening for Therapeutic Agents for Treating a Hematological Malignancy

Provided by the invention are methods for identifying therapeutic agentsfor treating multiple myeloma or another hematological malignancy thatare based on the binding of cyclophilin A to CD147 on multiple myelomacells.

For example, a method for identifying an agent for treating multiplemyeloma (MM) or other hematological malignancies includes providing afirst polypeptide comprising a CD147 polypeptide and a secondpolypeptide comprising an extracellular cyclophilin A (eCyPA)polypeptide sequence under conditions that allow for binding of theCD147 polypeptide and the eCyPA sequence to form a complex. The complexis then contacted with a test agent, and the complex is assayed todetermine whether the test agent disrupts binding of the first andsecond polypeptide. Disruption of the binding of the first polypeptideand second polypeptide by the test agent indicates the test agent is apotential therapeutic agent for treating MM.

A test agent that disrupts CD147 binding to eCyPA can be furthercharacterized to determine its suitability as a therapeutic agent fortreating MM. For example, a promising test agent can be used asdescribed in the examples below to determine whether it inhibitsproliferation of an MM cell population and/or whether it inhibitsmigration of MM cells into bone marrow Inhibition of MM cellproliferation and/or migration indicates the test agent is a therapeuticagent for treating MM.

The first and second polypeptide sequences can be CD147 polypeptidesequences and cyclophilin A sequences known in the art. Thus, in someembodiments, extracellular cyclophilin A polypeptide sequences includethe following amino acid sequence:

(SEQ ID NO: 1) GGSMVNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGEKGFGYKGSCFHRIIPGFMCQGGDFTRHNGTGGKSIYGEKFEDENILKHTGPGILSMANAGPNTNGSQFFICTAKTEWLDGKVVFGKVKEGMNIVEAMERFGSRN GKTSKKITIADCGQLE

In some embodiments the CD147 polypeptide sequences include apolypeptide with the following amino acid sequence of a human CD147polypeptide:

(SEQ ID NO: 2) MAAALFVLLGFALLGTHGASGAAGFVQAPLSQQRWVGGSVELHCEAVGSPVPEIQWWFEGQGPNDTCSQLWDGARLDRVHIHATYHQHAASTISIDTLVEEDTGTYECRASNDPDRNHLTRAPRVKWVRAQAVVLVLEPGTVFTTVEDLGSKILLTCSLNDSATEVTGHRWLKGGVVLKEDALPGQKTEFKVDSDDQWGEYSCVFLPEPMGTANIQLHGPPRVKAVKSSEHINEGETAMLVCKSESVPPVTDWAWYKITDSEDKALMNGSESRFFVSSSQGRSELHIENLNMEADPGQYRCNGTSSKGSDQAIITLRVRSHLAALWPFLGIVAEVLVLVTIIFIYEKRRKPEDVLDDDDAGSAPLKSSGQHQNDKGKNVRQRNSSTest Agents

The term “test agent” or “test compound” refers to any chemical entity,pharmaceutical, drug, and the like, that can be used to treat or preventa disease, illness, sickness, or disorder of bodily function, orotherwise alter the physiological or cellular status of a sample (e.g.,the level of disregulation of apoptosis in a cell or tissue). Testagents comprise both known and potential therapeutic compounds. A testcompound can be determined to be therapeutic by using the screeningmethods of the present invention.

A “known therapeutic compound” refers to a therapeutic compound that hasbeen shown (e.g., through animal trials or prior experience withadministration to humans) to be effective in such treatment orprevention.

The screening methods can include those known or used in the art orthose first described herein. For example, in one embodiment a CD147 isimmobilized on a microtiter plate and incubated with cyclophilin A inthe presence of a test agent. Subsequently, the complex can be detectedusing a secondary antibody, and absorbance can be detected on a platereader.

The test agent can be a small molecule or a large molecule. A “smallmolecule” as used herein, is meant to refer to a composition that has amolecular weight of less than about 5 kD and most preferably less thanabout 4 kD. Small molecules can be, e.g., nucleic acids, peptides,polypeptides, peptidomimetics carbohydrates, lipids or other organic orinorganic molecules. Libraries of chemical and/or biological mixtures,such as fungal, bacterial, or algal extracts, are known in the art andcan be screened with any of the assays of the invention. Examples ofmethods for the synthesis of molecular libraries can be found in theart, for example in: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. U.S.A.90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci. U.S.A. 91: 11422;Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho, et al., 1993.Science 261: 1303; Carrell, et al., 1994. Angew. Chem. Int. Ed. Engl.33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061;and Gallop, et al., 1994. J. Med. Chem. 37: 1233.

The test agent need not be any particular structure or size. In someembodiments, the test agent is a nucleic acid, a polypeptide, a smallmolecule or combinations thereof, an inhibitory nucleic acid, e.g., atriplex forming oligonucleotide, an aptamer, a ribozyme, an antisenseRNA, a short interfering RNA (siRNA), or a micro-RNA (miRNA).

In some embodiments, the polypeptide is a polypeptide binding partner ofa cyclophilin A molecule or CD147 molecule, e.g., an antibody, e.g., ananti-CyPA antibody. Anti-CyPA antibodies for treating HIV infection aredescribed in, e.g., U.S. Pat. No. 5,840,305.

Antibodies are preferably modified to reduce the likelihood of anunwanted host reaction. One example of such a modification is ahumanized antibody. Humanized forms of non-human (e.g., murine)antibodies are chimeric molecules of immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′) or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species.

Human antibodies can also be produced using other techniques known inthe art, including phage display libraries (Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)). The techniques of Cole and Boerner are also available for thepreparation of human monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner etal., J. Immunol., 147(1):86-95 (1991)). Similarly, human antibodies canbe made by introduction of human immunoglobulin loci into transgenicanimals, e.g., mice in which the endogenous immunoglobulin genes havebeen partially or completely inactivated. Upon challenge, human antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly, and antibodyrepertoire. This approach is described, for example, in U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and inthe following scientific publications: Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison,Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14,845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); andLonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

In addition, cyclophilin A antibodies and anti-CD147 antibodies can beused to create a therapeutic agent for treating MM, or another diseaseby adapting methods for making humanized antibodies described in U.S.Pat. No. 8,673,593. The method includes providing a DNA encoding thevariable domains of a donor CyPA antibody and determining the amino acidsequence of the CDR regions of the donor monoclonal antibody from theDNA, selecting human acceptor antibody sequences; and producing ahumanized CyPA antibody comprising the CDRs from the donor antibody andvariable region frameworks from the human acceptor antibody sequences.If desired, the method can further include determining whether humanizedantibody disrupts binding of first polypeptide comprising a CD147polypeptide and a second polypeptide comprising an extracellularcyclophilin A (eCyPA) polypeptide sequence under conditions that allowfor binding of the CD147 polypeptide and the eCyPA sequence. Disruptionof the binding of the first polypeptide and second polypeptide by thehumanized antibody indicates it is a therapeutic agent for treating MM.

The antibody preferably binds specifically (or selectively) to either acyclophilin A or CD147 molecule. The phrase “specifically (orselectively) binds” to an antibody or “specifically (or selectively)immunoreactive with,” when referring to a protein or peptide, refers toa binding reaction that is determinative of the presence of the proteinin a heterogeneous population of proteins and other biologics. Thus,under designated immunoassay conditions, the specified antibodies bindto a particular protein at least two times the background and do notsubstantially bind in a significant amount to other proteins present inthe sample. Specific binding to an antibody under such conditions mayrequire an antibody that is selected for its specificity for aparticular protein. A variety of immunoassay formats may be used toselect antibodies specifically immunoreactive with a particular protein.For example, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Antibodies, A Laboratory Manual (1988), for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity). Typically a specific or selective reactionwill be at least twice background signal or noise and more typicallymore than 10 to 100 times background.

If desired, the antibody can be provided conjugated or coupled to adetectable label, a radioactive label, an enzyme, a fluorescent label, aluminescent label, a bioluminescent label, or a therapeutic agent.

Detecting eCyPA and CD147 Complexes

The first and second polypeptide can be provided in either a cell-freeor a cell-based system. Art recognized methods for measuringprotein-protein interactions can be used to characterize binding of theeCyPA sequence and CD147 polypeptide in the presence of the test agent,for example, by coupling the test agent with a radioisotope or enzymaticlabel such that binding of the test compound to the antigen orbiologically-active portion thereof can be determined by detecting thelabeled compound in a complex. For example, test agents can be labeledwith ¹²⁵l, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and theradioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, test agents can beenzymatically-labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

In one embodiment, the assay comprises contacting eCyPA sequence-CD147polypeptide complex with a test agent, and determining the ability ofthe test compound to interact with the complex or otherwise disrupt theexisting complex. In this embodiment, determining the ability of thetest compound comprises determining the ability of the test compound topreferentially bind to the eCyPA sequence and/or the CD147 or abiologically-active portion thereof, as compared to its binding partner.

Observation of the complex in the presence and absence of a test agentcan be accomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein that adds adomain that allows one or both of the proteins to be bound to a matrixcan be provided. In one embodiment, GST-antibody fusion proteins orGST-antigen fusion proteins are adsorbed onto glutathione Sepharose®beads (Sigma Chemical, St. Louis, Mo.) or glutathione-derivatizedmicrotiter plates, that are then combined with the test compound, andthe mixture is incubated under conditions conducive to complex formation(e.g., at physiological conditions for salt and pH).

Following incubation, the beads or microtiter plate wells are washed toremove any unbound components, the matrix immobilized in the case ofbeads, complex determined either directly or indirectly. Alternatively,the complexes can be dissociated from the matrix, and the level ofantibody-antigen complex formation can be determined using standardtechniques.

In some embodiments, one of either the eCyPA sequence or the CD147sequence is immobilized to facilitate separation of complexed fromuncomplexed forms of one or both following introduction of the restagent, as well as to accommodate automation of the assay. If desired,either member of the putative complex can be immobilized utilizingbiotin and streptavidin. Biotinylated antibody or antigen molecules canbe prepared from biotin-NHS (N-hydroxysuccinimide) using techniqueswell-known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical). Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using such other antibodiesreactive with the antibody or antigen.

In some embodiments, binding of the test agent to the complex isdetected using assay AlphaScreen® technology (PerkinElmer, Waltham,Mass.).

Cells Used in Screening Assays

When cell-based assays are used, CD-147 expressing cells can be providedby any cell (e.g., MM1S, OPM1, H929, U266 cells) that expresses CD147 inamounts sufficient to detectably bind an eCyPA polypeptide orCD147-binding fragment of an eCyPA polypeptide. Cells can be prokaryoticor eukaryotic cells. The cell can be provided in vitro or in vivo. Cellscan be prokaryotic or eukaryotic, for example, mammalian cells includingboth human and non-human mammalian cells (e.g., a rodent such as a mouseor rat cell).

Cells can be primary cells or established cell lines. In someembodiments hematopoietic cells are used. The term “hemapoietic cells”as used herein includes all blood cell types, including those from themyeloid lineage (monocytes and macrophages, neutrophils, basophils,eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells),and lymphoid lineages (T-cells, B-cells, NK-cells).

In some embodiments, the methods described herein are performed on cellsamples. A “sample” in the context of screening assays is understoodwithin the scope of the invention to refer to a suitable cell, group ofcells, animal model or human. These samples do not have to be derivedfrom a subject. A sample in this context can be a group of cells from acell line. Preferably cell lines are derived from a hematopoieticdisorder. A non-limiting list of samples includes bone marrow aspiratesor bone marrow biopsy (for myeloma, leukemias and other hematopoieticdisorders), lymph node samples lymphomas and other hematopoieticdisorders) and peripheral blood samples (for leukemias and otherhematopoietic disorders). The sample may be of a particular type ofhematopoietic cell, for example a population of B lymphocytes and/or Tlymphocytes.

The presence and/or level of proteins used in the methods describedherein (e.g., eCyPA protein and CD147 polypeptides) can be evaluatedusing methods known in the art, e.g., using quantitative immunoassaymethods such as enzyme linked immunosorbent assays (ELISA), see above,immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA),radioimmunoassay (RIA), and Western blot analysis.

Similarly, the presence and/or level of transcripts encoding theseproteins can be evaluated using RNA detection methods known in the art,e.g., quantitative transcription detection such as a transcription-basedamplification system (TAS). Some examples of RNA detection systemsinclude PCR and QRT-PCR-based amplification systems, ligase chainreaction, Qβ, replicase, reverse transcriptase-coupled nucleic acidsequence-based amplification (NASBA), self-sustained sequencereplication (3SR), strand displacement amplification (SDA), or reversetranscriptase-coupled rolling circle amplification (RCA).

Diagnosing and Determining a Prognosis for a Subject with MultipleMyeloma

The link identified by the inventors between eCyPA and multiple myelomaalso provides new methods for diagnosing and otherwise assessing MM in asubject. Multiple myeloma (MM) or a multiple myeloma precursor conditionin a subject can be diagnosed by providing a sample from a subject andassaying the sample to determine a level of eCyPA in the sample toobtain an eCyPA test value. The test value is compared to an eCyPAreference value calculated for a sample whose MM status is known. AneCyPA test value greater than a reference eCyPA value in a referencesample known not to have MM indicates that the subject has MM or a MMprecursor conditions. Conversely, an eCyPA test value equal to or lessthan a reference eCyPA value in a sample known to have MM indicates thatthe subject does not have multiple myeloma.

Similarly, the prognosis for a subject with multiple myeloma (MM) can bedetermined by providing a sample from a subject with MM and assaying thesample to determine a level of eCyPA in the sample to obtain an eCyPAtest value, then comparing the eCyPA test value to an eCyPA referencevalue calculated for a sample whose MM status is known. An eCyPA testvalue greater than a reference eCyPA value in a sample known not to haveMM indicates that the subject has a poor prognosis, while an eCyPA testvalue less than a reference eCyPA value in a sample known to have MMindicates that the subject has a good prognosis.

Efficaciousness of a treatment for multiple myeloma can be determined byproviding a sample from a subject with MM and assaying the sample todetermine a level of eCyPA in the sample to obtain an eCyPA test value.The eCyPA test value is compared to an eCyPA reference value calculatedfor a sample whose MM status is known. An eCyPA test value greater thana reference eCyPA value in a reference sample known not to have MMindicates that the treatment is not efficacious, and an eCyPA test valueless than a reference eCyPA value in a sample known to have MM indicatesthat the treatment is efficacious. In some embodiments, the referencesample is obtained from the same subject prior to beginning treatment ofMM or at an earlier point in treatment of MM.

The progression of multiple myeloma (MM) in a subject can similarly bedetermined; an eCyPA test value greater than a reference eCyPA value ina sample known not to have MM indicates that the subject has a MM moreadvanced than MM in subjects from which the reference eCyPA value iscalculated, while an eCyPA test value less than the reference eCyPAvalue indicates that the subject has a MM less advanced than MM insubjects from which the reference eCyPA value is calculated.

The threshold for determining how a test sample is scored in the assaysdescribed herein, e.g., whether a test sample is scored positive, can bealtered depending on the sensitivity or specificity desired. Theclinical parameters of sensitivity, specificity, negative predictivevalue, positive predictive value and efficiency are typically calculatedusing true positives, false positives, false negatives and truenegatives. A “true positive” sample is a sample that is positiveaccording to an art recognized method, which is also diagnosed aspositive (high risk for early attack) according to a method of theinvention. A “false positive” sample is a sample negative by anart-recognized method, which is diagnosed positive (high risk for earlyattack) according to a method of the invention. Similarly, a “falsenegative” is a sample positive for an art-recognized analysis, which isdiagnosed negative according to a method of the invention. A “truenegative” is a sample negative for the assessed trait by anart-recognized method, and also negative according to a method of theinvention. See, for example, Mousy (Ed.), Intuitive Biostatistics NewYork: Oxford University Press (1995), which is incorporated herein byreference.

As used herein, the term “sensitivity” means the probability that alaboratory method is positive in the presence of the measured trait.Sensitivity is calculated as the number of true positive results dividedby the sum of the true positives and false negatives. Sensitivityessentially is a measure of how well a method correctly identifies thosewith disease. For example, cut-off values can be selected such that thesensitivity of diagnosing an individual is at least about 60%, and canbe, for example, at least about 50%, 65%, 70%, 75%, 80%, 85%, 90% or95%.

As used herein, the term “specificity” means the probability that amethod is negative in the absence of the measured trait. Specificity iscalculated as the number of true negative results divided by the sum ofthe true negatives and false positives. Specificity essentially is ameasure of how well a method excludes those who do not have the measuredtrait. For example, cutoff values can be selected so that when thesensitivity is at least about 70%, the specificity of diagnosing anindividual is in the range of 30-60%, for example, 35-60%, 40-60%,45-60% or 50-60%.

The term “positive predictive value,” as used herein, is synonymous with“PPV” and means the probability that an individual diagnosed as havingthe measured trait actually has the disease. Positive predictive valuecan be calculated as the number of true positives divided by the sum ofthe true positives and false positives. Positive predictive value isdetermined by the characteristics of the diagnostic method as well asthe prevalence of the disease in the population analyzed. The cut-offvalues can be selected such that the positive predictive value of themethod in a population having a disease prevalence of 15% is at leastabout 5%, and can be, for example, at least about 8%, 10%, 15%, 20%,25%, 30% or 40%.

As used herein, the term “efficiency” means the accuracy with which amethod diagnoses a disease state. Efficiency is calculated as the sum ofthe true positives and true negatives divided by the total number ofsample results, and is affected by the prevalence of the trait in thepopulation analyzed. The cut-off values can be selected such that theefficiency of a method of the invention in a patient population having aprevalence of 15% is at least about 45%, and can be, for example, atleast about 50%, 55% or 60%.

For determination of the cut-off level, receiver operatingcharacteristic (ROC) curve analysis can be used. In some embodiments,the cut-off value for the classifier can be determined as the value thatprovides specificity of at least 90%, at least 80% or at least 70%.

Treating Subjects with High Levels of eCyPA

Subjects determined to have multiple myeloma, a multiple myeloma relatedcondition, or inflammation based on elevated levels of eCyPA can betreated using one or more treatment modalities known in the art. Forexample, treatment multiple myeloma, a multiple myeloma relatedcondition can be with a chemotherapeutic agent, radiation agent,hormonal agent, biological agent, an anti-inflammatory agent, or acombination of two or more of these agents.

Chemotherapeutic agents include, e.g., sanglifehrin A,sarcosine-3(4-methylbenzoate) (SmBz), voclosporin, cyclosporin A,NVP018, alisporivir, NIM811, MMM284, CD147 antibody, CyPA antibody,tamoxifen, trastuzamab, raloxifene, doxorubicin, fluorouracil/5-fu,pamidronate disodium, anastrozole, exemestane, cyclophosphamide,epirubicin, letrozole, toremifene, fulvestrant, fluoxymester one,trastuzumab, methotrexate, megastrol acetate, docetaxel, paclitaxel,testolactone, aziridine, vinblastine, capecitabine, goselerin acetate,zoledronic acid, taxol, vincristine, and/or HDAC/TDAC inhibitors andaggresome inhibitors disclosed in U.S. Pat. No. 8,999,289. Additionaltreatment strategies include, e.g., autologous stem cell transplantationand allogeneic effector cell transplantation, to develop an effectivetreatment strategy based on the stage of myeloma being treated (see,e.g., Multiple Myeloma Research Foundation, Multiple Myeloma: Stem CellTransplantation 1-30 (2004); U.S. Pat. Nos. 6,143,292, and 5,928,639,Igarashi, et al. Blood 2004, 104(1): 170-177, Maloney, et al. 2003,Blood, 102(9): 3447-3454, Badros, et al. 2002, J. Clin. Oncol.,20:1295-1303, Tricot, et al. 1996, Blood, 87(3):1196-1198; the contentsof which are incorporated herein by reference).

The effectiveness of a multiple myeloma diagnosis or prognosis usingeCyPa levels can be compared to other methods known in the art forassessing multiple myeloma or a related condition. The multiple myelomastaging system most widely used since 1975 has been the Durie-Salmonsystem, in which the clinical stage of disease (Stage I, II, or III) isbased on four measurements (see, e.g., Durie and Salmon, 1975, Cancer,36:842-854). These four measurements are: (1) levels of monoclonal (M)protein (also known as paraprotein) in the serum and/or the urine; (2)the number of lytic bone lesions; (3) hemoglobin values; and, (4) serumcalcium levels. These three stages can be further divided according torenal function, classified as A (relatively normal renal function, serumcreatinine value<2.0 mg/dL) and B (abnormal renal function, creatininevalue.gtoreq.2.0 mg/dL). A new, simpler alternative is the InternationalStaging System (ISS) (see, e.g., Greipp et al., 2003, “Development of aninternational prognostic index (IPI) for myeloma: report of theinternational myeloma working group”, The Hematology). The ISS is basedon the assessment of two blood test results, beta₂-microglobulin (β₂-M)and albumin, which separates patients into three prognostic groupsirrespective of type of therapy.

Administration of the pharmaceutical compositions at selected dosageranges and routes typically elicits a beneficial response as defined bythe European Group for Blood and Marrow transplantation (EBMT) in Table1, below (taken from U.S. Pat. No. 8,632,772).

TABLE 1 lists the EBMT criteria for response: EBMT/IBMTR/ABMTR¹ Criteriafor Response Complete No M-protein detected in serum or urine byResponse immunofixation for a minimum of 6 weeks and fewer than 5%plasma cells in bone marrow Partial >50% reduction in serum M-proteinlevel Response and/or 90% reduction in urine free light chain excretionor reduction to <200 mg/24 hrs for 6 weeks² Minimal 25-49% reduction inserum M-protein level Response and/or 50-89% reduction in urine freelight chain excretion which still exceeds 200 mg/24 hrs for 6 weeks³ NoChange Not meeting the criteria or either minimal response orprogressive disease Plateau No evidence of continuing myeloma-relatedorgan or tissue damage, <25% change in M- protein levels and light chainexcretion for 3 months Progressive Myeloma-related organ or tissuedamage Disease continuing despite therapy or its reappearance in plateauphase, >25% increase in serum M- protein level (>5 g/L) and/or >25%increase in urine M-protein level (>200 mg/24 hrs) and/or >25% increasein bone marrow plasma cells (at least 10% in absolute terms)² RelapseReappearance of disease in patients previously in complete response,including detection of paraprotein by immunofixation ¹EBMT: EuropeanGroup for Blood and Marrow transplantation; IBMTR: International BoneMarrow Transplant Registry; ABMTR: Autologous Blood and MarrowTransplant Registry.

Additional criteria that can be used to measure the outcome of atreatment include “near complete response” and “very good partialresponse”. A “near complete response” is defined as the criteria for a“complete response” (CR), but with a positive immunofixation test. A“very good partial response” is defined as a greater than 90% decreasein M protein (see, e.g., Multiple Myeloma Research Foundation, MultipleMyeloma: Treatment Overview 9 (2005)).

The degree to which administration of the composition elicits a responsein an individual clinically manifesting at least one symptom associatedwith MM, depends in part, on the severity of disease, e.g., Stage I, II,or III, and in part, on whether the patient is newly diagnosed or haslate stage refractory MM. Thus, in some embodiments, administration ofthe pharmaceutical composition elicits a complete response.

In some embodiments, administration of the pharmaceutical compositionelicits a very good partial response or a partial response. In otherembodiments, administration of the pharmaceutical composition elicits aminimal response. In other embodiments, administration of thepharmaceutical composition prevents the disease from progressing,resulting in a response classified as “no change” or “plateau” by theEBMT.

Computer Implemented Embodiments

Information from the eCyPA levels and other test results can implementedin computer programs executed on programmable computers that include,inter alia, a processor, a data storage system (including volatile andnon-volatile memory and/or storage elements), at least one input device,and at least one output device. Program code can be applied to inputdata to perform the functions described above and generate outputinformation. The output information can be applied to one or more outputdevices, according to methods known in the art. The computer may be, forexample, a personal computer, microcomputer, or workstation ofconventional design.

In some embodiments, the a machine-readable storage medium can comprisea data storage material encoded with machine readable data or dataarrays which, when using a machine programmed with instructions forusing the data, is capable of use for a variety of purposes, such as,without limitation, subject information relating to a diagnosing a typeor subtype of ovarian cancer, evaluating the effectiveness of atreatment (e.g., surgery or chemotherapy).

Each program can be implemented in a high level procedural or objectoriented programming language to communicate with a computer system.However, the programs can be implemented in assembly or machinelanguage, if desired. The language can be a compiled or interpretedlanguage. Each such computer program can be stored on a storage media ordevice (e.g., ROM or magnetic diskette or others as defined elsewhere inthis disclosure) readable by a general or special purpose programmablecomputer, for configuring and operating the computer when the storagemedia or device is read by the computer to perform the proceduresdescribed herein.

The health-related data management system of the invention may also beconsidered to be implemented as a computer-readable storage medium,configured with a computer program, where the storage medium soconfigured causes a computer to operate in a specific and predefinedmanner to perform various functions described herein.

Bone Marrow Scaffolding Substrates

Also provided by the invention is a composition that includes abiocompatible substrate and bone marrow-derived endothelial cells (BMEC)admixed with a biocompatible substrate. The BMEC cells express eCyPA inan amount sufficient to stimulate proliferation or migration of multiplemyeloma (MM) cells. In some embodiments, the substrate is apoly-caprolactone scaffolding (PCLS). To admix the BMEC with abiocompatible substrate, the PCLS is pre-coated with fibronectin.

Other Malignancies and Conditions

While illustrated with multiple myeloma cells, the methods andcompositions described herein can be adapted for any malignancy in whichincreased cyclophilin A levels or activity promotes proliferation and/ormigration of malignant cells to bone marrow, and/or is associated withCD147 expression. This includes chronic lymphocytic leukemia (CLL) andlymphoplasmacytic lymphoma (LPL) (see examples below showing eCyPAaffects these cells in ways similar to its effects on MM cells).

Additional conditions include precursor conditions or other conditionsrelated to MM, such as Monoclonal Gammopathy of UndeterminedSignificance (MGUS), smoldering myeloma, asymptomatic MM, andsymptomatic MM, ranging from newly diagnosed to late stagerelapsed/refractory.

Additional non-limiting examples of hematopoietic cancers for whichmethods of the invention can be used include leukemias (myeloid andlymphoid types), and lymphomas of B- and T-cell lineages), polycythermisvera and other myeloproliferative diseases. Examples of leukemiasinclude acute lymphoblastic leukemia (ALL), adult T cell leukemia (ATL),acute myeloblastic leukemia (AML) and chronic myeloid leukemia (CML).The method can also be used to treat carcinomas that frequentlymetastasize to the bone marrow such as breast and prostate cancers.

In addition, the methods disclosed herein are useful in treatinginflammation generally. The screening methods are useful for identifyingnew therapeutic agents for treating inflammation. Similarly, the methodsdiscussed above can be used in diagnosing, prognosis, efficaciousnessand assessing the progression of inflammation in a subject.

The invention will be further illustrated in the following non-limitingexamples. The examples show that B-cell malignancies frequently colonizethe bone marrow (BM). New insights into the pathogenesis of thesemalignancies suggest that cells in the BM microenvironment play acritical role. Using multiple myeloma (MM) as a model of a terminallydifferentiated B-cell neoplasm that selectively colonizes the BM, andtaking advantage of a broadly adaptable mouse xenograft model systememploying bone-like scaffolds coated with human BM-derived cells, wedemonstrated that BM-derived endothelial cells (BMECs), but notBM-derived stromal cells (BMSCs), secrete cyclophilin A (eCyPA), anextracellular pleiotropic signaling factor that promotes migration,growth, and BM colonization of MM cells via binding to its cognatereceptor, CD147, on MM cells. The clinical/translational implications ofthis work are highlighted by the observation of significantly highereCyPA levels in BM serum than in peripheral blood (PB) of the same MMpatient, and that blockade of the eCyPA/CD17 axis supresses BM-homingand tumor growth in a mouse xenograft model of MM. Furthermore, eCyPAalso promotes cell migration in chronic lymphocytic lymphoma andlymphoplasmacytic lymphoma, two other B-cell malignancies that colonizethe BM and express CD147. Taken together, these findings provideevidence of a possible link between chronic inflammation, immunedysregulation, and the pathogenesis of these B-cell malignancies.Importantly, our findings also offer a compelling rationale fortargeting the eCPA/CD147 signaling complex as a novel treatment approachfor these malignancies.

The examples offer experimental, as well as clinical and translational,evidence of a novel molecular mechanism for the migration and BM homingof MM cells. We document that eCyPA, but not its homolog CyPB, isproduced in (FIG. 4H) and secreted by (FIG. 4E) BMECs, but notendothelial cells from other vascular beds (FIG. 12A) or by otherstromal cells in the BM (FIG. 4G). eCyPA functions as a pleiotropicsignaling factor (FIG. 5C) that promotes migration (FIG. 5A), growth(FIG. 5F), and BM colonization of MM cells (FIGS. 5G-I) via binding toits cognate receptor, CD147, on MM cells (FIG. 6). In the process, wehave identified eCyPA as a novel target of the β-catenin/BCL9transcriptional complex (FIG. 10I), and have provided evidence for anovel functional role of the Wnt signaling pathway and of BMECs intrafficking, recruitment, growth, and progression of MM (FIGS. 1-3). Inaddition, we have identified BMECs as the main source of eCyPA, but notSDF-1 which is secreted by other stromal cells (FIG. 14I). Thus ourfindings provide the first indication that BMECs and BMSCs playdifferent roles in MM pathogenesis, and do so via different molecularmechanisms.

Although CyPA was initially recognized as the host cell receptor for thepotent immunosuppressive drug Cyclosporine A, recent studies haverevealed that it can be secreted by cells in response to inflammatorystimuli. Secreted eCyPA can initiate a signaling response in targetcells and is a potent neutrophil, eosinophil, and T cell chemoattractant31-34. Studies aimed at establishing the mechanism whereby eCyPAmediates its chemotactic activity have identified CD147 as the principalsignaling receptor for eCyPA. Indeed, all human 37 and mouse 32leucocytes examined to date require CD147 expression for eCyPA-mediatedchemotaxis to occur.

CD147, also known as Extracellular Matrix Metalloproteinase Inducer(EMPRIN), is a type I integral transmembrane glycoprotein that belongsto the immunoglobulin super family. It plays critical roles inintercellular communication involved in chronic inflammation,immune-related functions, tumor metastasis and angiogenesis, and HIVinfection. CD147 induces expression of the matrix metallopeptidasesrequired for tumor invasion and metastasis via cell-cell and cell-matrixinteractions. Recently, expression of CD147 has been broadly correlatedwith progression in ovarian, hepatocellular, bladder, cervical, lung,and gallbladder carcinomas, as well as hematological malignancies suchas MM.

In regard to MM, we show that CyPB, although tested at much higherconcentrations than those in BM serum of MM patients (FIG. 4J), inducesproliferation of MM cells. Of particular relevance to our studies,however, is the observation that CD147 is not expressed in normal plasmacells, yet its expression correlates with MM progression.

Example 1 General Materials and Methods

Patient Samples and Cell Lines

BM specimens were obtained from patients with MM or from normal donorsin accordance with Dana-Farber Cancer Institute Review Board protocols,and with informed consent in compliance with the Helsinki Declaration.BM mononuclear cells were isolated with the aid of a Ficoll gradient,and BMECs as well as MM primary cells were isolated using CD34 or CD138magnetic beads (Miltenyi Biotec, Auburn, Calif.), respectively, asdescribed in Sukhedo et al., Proc. Nat. Acad. Sci. (USA) 104:7516-7521(2007). Primary BMECs and primary BMSCs from the same MM patient wereprepared as follows: after Ficoll gradient, BM mononuclear cells weretreated with collagenase, and single-cell suspensions were placed onplastic dishes. When only attached cells remained in the cultures, theywere divided into two fractions and one was stored without furtherprocessing for subsequent use as BMSCs. From the other fraction, wepurified BMECs using CD34 immunomagnetic beads. Purified BMECs cellswere further expanded in culture ex-vivo and their identity confirmed byFC and immunoblot analysis using several endothelial cell markers (CD31,VEGF R1, CD138, and Factor VIII). Primary CLL and LPL cells werepurified using CD19 magnetic beads (Miltenyi Biotec, Auburn, Calif.).All primary cells were >90% pure and fresh for functional studies orstored in liquid nitrogen for other subsequent studies. Established MMcell lines (MM1S, ABNL6, U266, H929, OPM2, and RPMI) were all from theCarrasco laboratory. The HG3 (CLL) and BCWM.1 (LPL) cells were providedby Drs. Wu and Treon, respectively. Human BM-derived endothelial celllines BMEC-60 and HBME-1 were kindly provided by Drs. van der Schoot andGiuliani, respectively. The stromal cell line HS5 was obtained from Dr.Mitsiades's lab. All cells were grown at 37° C. under a 5% CO₂humidified atmosphere in RPMI 1640 medium supplemented with 10% fetalbovine serum (FBS). All cell lines were routinely tested using HumanCell Line Genotyping System (Promega), and were periodically surveyedfor mycoplasma contamination with the help of a commercial detection kit(LT07-218, Lonza). Serum from BM and PB of MM patients were obtainedfrom the Jerome Lipper Multiple Myeloma Center, Dana-Farber CancerInstitute. Studies with human subjects were approved by the Dana-FarberCancer Institute Review Board Committee (IRB #01-206), and informedconsent was obtained from all subjects.

Scaffold Mouse Xenograft Model

2×10⁶ BMEC-60 cells, HS5 cells, PBMEC or PBMSC were trypsinized andseeded into poly-ε-caprolactone polymeric scaffolds generously providedby Dr. Tassone (see Calimeri et al., Leukemia 25: 707-711 (2011).Scaffolds were first coated with 50 μg/ml fibronectin (Santa Cruz). Theseeded scaffolds were cultured in vitro for 2 weeks and implanted s.c.in the right or left flank of five-week old non-γ-irradiatedCB17.Cg-PrkdcscidLystbg-J/Crl male mice (Charles River). Two weekslater, 5×10⁶ luciferase-labeled MM1S or H929 cells were injected via thetail-vein into scaffold-implanted mice. Tumor burden was analyzed on thebasis of luciferase bioluminescence using an LAS-4000 Luminescent ImagerAnalyzer (Fujifilm). Mice were euthanized 4 weeks later and thescaffolds removed for histological and IHC examination as described inMani et al., Cancer Res.69: 7577-86, 2009. Experiments were repeatedindependently three times. Animal experiments were approved by theDana-Farber Cancer Institute Institutional Animal Care and Use Committee(IRB#10-039) and were in compliance with ethical practice. All theexperiments were performed blindly, as the investigator who implantedthe mouse did not know the content of the scaffolds. In animal studiesusing the Xenograft scaffold system, one experiment was excluded becauseMM1S-luc or H929-luc cells grew within both the BMEC-60 coated scaffoldand the spine and skull of the mouse, precluding optimal evaluationusing the Xenogen system.

For in vivo treatment with CD147 Ab, the mouse scaffold system wasmodified as follows: scaffolds were pre-coated with BMEC-60 cells,incubated with MM1S-luc cells, and then implanted into the flank ofnon-γ-irradiated CB17.Cg-PrkdcscidLystbg-J/Crl mice. One weekpost-implantation the mice were subjected to whole-body imaging, andthose with a comparable tumor burden were selected for Ab therapy. Micewere injected with 100 μl of a solution containing either 100 ng ofisotype Ab or anti-CD147 Ab sc every other day in a region adjoing thescaffold. Tumor growth within each scaffold was evaluated by Xenogenimaging every five days. After 15 days, the scaffolds were removed andprocessed for histological and IHC analysis.

Complement-Dependent Cytotoxicity (CDC)

MM.1S-Luc cells (1×10⁶) were labeled with Calcein-AM (1 μg/ml,Technologies, Grand Island, N.Y.) for 30 min in a CO₂ incubator at 37°C., washed twice with fresh mouse medium, and then cultured for 1 h withanti-CD147 Ab or isotype control Ab (10 μg/ml), in the absence orpresence of mouse serum (20% final concentration). Cells were spun downand fluorescence in the supernatant was measured using a 490 nmexcitation filter and a 520 nm emission filter. Percent specific lysiswas calculated from the formula % Lysis=(Sample RFU−Background RFU/TotalRFU−Background RFU)×100.

Immunoblot Analysis

Total protein samples were prepared from non-attached H929 and MM1Scells grown in CM from cultures of BMEC-60 or BMEC-1 cells attached for72 hr. In some experiments, the cells were co-cultured with BMEC-60cells using transwell chambers (Corning Costar, 0.45 μm pore diameter).Immunoblotting was performed as described in Takada et al., Sci. Transl.Med. 4, 148ra117 (2012). When MM1S and H929 cells were treated witheCyPA or CD147 Ab, they were first incubated with eCyPA or CD147 Ab for5 hrs in serum-free medium. Then 0.5% serum was added, and incubationwas resumed for another 72 hrs before performing protein preparation.Anti-human primary antibodies included: BCL9 (2D4, Abnova), CyPA(ERPR7511, Abcam), CD147 (#13287, Cell Signaling), pSTAT3 (#9131, CellSignaling), pERK (#4370, Cell Signaling), PARP (#9542, Cell Signaling),pAKT (#9271, Cell Signaling), Active-β-catenin (05-665, Millipore),Factor VIII (A0082, DAKO), MMP-9 (MAB911, R&D), CyPB (MAB5410, R&D), andhorseradish peroxidase(HRP)-conjugated actin (C-11, Santa Cruz). ActinAb was used as a loading control. Secondary antibodies included:anti-rabbit IgG (HRP)-conjugated (W402b, Promega), and anti-mouse IgG(HRP)-conjugated (W401b, Promega). Protein concentrations were measuredin triplicate by Bradford assay (BioRad). Optimal antibodyconcentrations were used according to the manufacturer's recomendations.

Lentiviral Infection

Validated expression plasmids for pLKO-shCyPA: #1, CCGGGTTCCTGCTTTCACAGAATTACTCGAGTAATTCTGTGAAAGCAGGAACTTTTTG (SEQ ID NO:3); #2,CCGGCTGACTGTGGACAACTCGAATCTCGAGATTCGAGTTGTCCACAGTCA GTTTTTG (SEQ IDNO:4) and pLKO-shCD147: #1, CCGGCCAGAATGACAAAGGCAAGAACTCGAGT (SEQ IDNO:5) TCTTGCCTTTGTCATTCTGGTTTTT (SEQ ID NO:6); #2,CCGGACAGTCTTCACTACCGTAGAACTCGAGTTCTACGGTAGTGAAGACTGTT TTTTG (SEQ IDNO:7) were purchased from Sigma. The pLKO-Control-shRNA and pLKO-shBCL9expression vectors are described in Mani et al., Cancer Res. 69:7577-7586 (2009). Lentiviral packaging and infection of MM1S-luc andBMEC-60 cells was done according to manufacturer's protocol (Sigma). Tominimize off-target effects of shRNAs, we implemented a series of assayconditions: i) shRNAs were designed to target the 3′UTR region, ii) theeffect of the target shRNAs had to differ from that of a control shRNAcontaining scrambled nucleotide sequences, iii) the phenotypic responsehad to be reproducible using two distinct target shRNAs (#1 and #2), andiv) identical results had to be obtained with more than one targetedcell.Cell Migration Assays

For cell migration assays, the top chamber of transwell plates (8 μMpore diameter, Corning Costar) was seeded with 2×10⁵ MM1S-luc cells, andthe bottom chamber was seeded with either medium (0.1% FBS) alone,BMEC-60 cells, BMEC-60 cell-conditioned medium, or plain mediumcontaining CyPA and/or CD147. After 12 hrs of incubation, MM1S-luc cellsthat had migrated to the bottom chamber were collected and quantified byXenogen Imaging. Migration of RPMI, U266, CLL, LPL, or primary tumorcells was evaluated by counting the number of unlabeled cells in thelower chamber. Each experiment was done in triplicate and repeatedtwice. Recombinant HPLC-purified eCyPA was purchased from BioMart Inc.and eCyPB (PPIB) human recombinant protein was purchased from Abnova. Inmost instances, 50 ng/ml of eCyPA or eCyPB diluted in serum-free RPMImedium was used. CM was collected from confluent cultures of BMEC-60cells incubated for 48 hours. When needed, the CM was treated with 100μg/ml proteinase K (19133, Qiagen) for 2 hrs and then deactivated for 15min at 70° C. Anti-CD147 (UM-8D6) antibody and monoclonal mouse IgG1(MOPC31C) were obtained from Ancell; anti-CXCR4 (MAB171) was from R&Dsystems. Migration data were normalized using the data obtained withmedium alone. Results are means±SD for triplicate assays.

Reporter Assays

Luciferase activity was measured using the Dual Luciferase ReporterAssay System (Promega), as described in Mani et al., Cancer Res. 69:7577-7586 (2009). To measure Wnt reporter activity, BMEC-60 cells weretransfected with TOP-FLASH, FOP-FLASH plasmid (Millipore), along with aninternal Renilla control plasmid (hRL-null). Transfection was performedusing FuGENE® (Roche) according to the manufacturer's protocol. Theresults were normalized to control for Renilla activity. The reporteddata represent the average of three independent transfection experimentsin triplicate.

Cell Proliferation and Viability Assays

Cell proliferation was evaluated by [³H]TdR incorporation as describedin Mani et al., Cancer Res. 69: 7577-7586 (2009). When proliferation ofMM cells was determined in the presence of HS5 or BMEC-60 cells, thelatter were previously gamma-irradiated (10,000 rads) and dispensed into96-well plates. Then, MM1S and H929 cells were co-cultured with eitherBMEC-60 cells, CM alone, CyPA, or CyPB for 72 hrs. Viability of MM1s-Luccells were co-cultured with BMEC-60 cells and then treated with drugs asdescribed was assessed, using the tumor cell-specific in vitrobioluminescence imaging (SS-BLI).

Histopathological and IHC Analysis

Tissue sections were processed as described in Mani et al., Cancer Res.69: 7577-7586 (2009). Human tissue samples were obtained from theDepartment of Pathology, Brigham and Women's Hospital. Sections wereincubated with primary antibodies (5 μg/ml) or the corresponding IgGfraction of pre-immune serum overnight at 4° C. in blocking solution (3%BSA/PBS). Anti-human primary specific antibodies included: BCL9(ab37305, Abcam), CD138 (PN IM 2757, Beckman Coulter), CD34 (M71165,DAKO), ERG (5115-1, Epitomics), Caspase-3 (#9664, Cell Signaling), CD147(MEM-M6/1, LifeSpan BioSciences), CyPA (ERPR7511, Abcam) and CyPB(AV44365, Sigma); and were visualized with the aid of the correspondingbiotinylated antibody coupled to streptavidin-peroxidase complex (VectorLabs). For CD147 immunohistochemistry, non-decalcified BM clots wereused. For negative controls, tissue sections were incubated in theabsence of primary antibodies or pre-immune serum from the species oforigin of the primary antibody. Optimal antibody concentrations wereused according to manufacturer's recommendation.

Immunofluorescence

Single-cell suspensions were spun onto glass slides using acytocentrifuge (Shandon) or were grown on polylysine-coated slides(p8920, Sigma), as described in Mani et al., Cancer Res. 69: 7577-7586(2009). Cells were fixed at room temperature in 2% paraformaldehyde for20 min, permeabilized in TBS-Tween 20 for 20 min, washed three times inPBS, and then blocked with 5% bovine serum albumin in PBS for 2 h beforeaddition of primary antibodies against BCL9 (ab37305, Abcam), β-catenin(CAT5-H10, Zymed), Myeloperoxidase (A0398, DAKO), CyPA (ERPR7511,Abcam), Alexa Fluor 647-conjugated CD147 (HIM6, Biolegend), orFITC-conjugated CD138 (MI15, Becton Dickinson). Cells were incubatedovernight with primary antibodies at 4° C., and then washed three timesin PBS before staining with secondary antibodies conjugated to AlexaFluor 488 (A11034, Molecular Probes) or Alexa Fluor 546 (A11035,Molecular Probes). Images were acquired with the aid of a Bio-RadRadiance 2000 laser scanning confocal or Nikon Eclipse E800phase-contrast microscope. Optimal antibody concentrations were usedaccording to manufacturer's recommendations.

Flow Cytometry (FC)

Harvested cells in aliquots of up to 1×10⁶ cells/100 μL were dispensedinto FACS tubes and stained for FC, as described Mani et al., CancerRes. 69: 7577-7586 (2009). Anti-human antibodies included: CD147-AlexaFluor 647 (HIM6, Biolegend), CD147-PE (8D12, eBioscience), CD19-FITC(SJ25C1, Becton Dickinson), CD19-APC (HIB19, Becton Dickinson), CD5-FITC(53-7.3, Becton Dickinson), CD138-APC (MI15, Becton Dickinson), andisotype control mouse IgG1

(P3.6.2.8.1, Bioscience). Optimal antibody concentrations were usedaccording to manufacturer's recommendations.

ELISA

Serum levels of eCyPA and eCyPB in BM and peripheral blood samples fromMM patients were measured by ELISA according to the manufacturer'sprotocols for eCyPA (KA1176, Abnova), eCyPB (ABIN414776, USCN LifeScience), and SDF-1 (DSA00, R&D), respectively. For the measurement ofeCyPA, eCyPB, and SDF-1 from cell supernatants, 1×10⁶ cells weredispensed in triplicate into 12-well plates and cultured for differenttimes. The spent medium was then replaced with an equal volume of freshmedium, the incubation was resumed for another 12 h, and finallytriplicate samples of supernatant containing equal amounts of totalprotein were used for ELISA analysis. Standard curves were linear, and100 ul of each sample was used for analysis in triplicate.

Mass Spectrometry (MS)

Bands excised from silver-stained gels were cut into approximately 1 mm³pieces, and the latter were analyzed by mass spectrometry, as describedin Peng et al., J. Mass Spectrom. 36: 1083-1091 (2001) and Levanon etal., Oncogene 29: 1103-1113 (2010). BMEC secretomes were analyzeddirectly, according to a previously described protocol (Ria et al.,Clin. Cancer Res. 15: 5369-5378 (2009)). Approximately 1×10⁶ BMEC-60cells were lentivirally transduced with control shRNA or shBCL9. These,along with HS5 cells, PBMEC #1, and PBMEC #2, were dispensed into12-well plates, which were kept for 6 h at 37° C. under 5% CO₂, andrinsed twice with 2 ml of PBS. Then, 200 μl of fresh PBS was added andthe cells were incubated at 37° C. under 5% CO₂ for another 24 h.Supernatants were collected and centrifuged for 5 min at 300 rpm toremove floating cells, and the amount of total protein in each samplewas measured. Total protein was loaded onto agarose gels in amountscommensurate with the decreased levels resulting from BCL9 knockdown;after electrophoresis, the gels were silver-stained according to themanufacturer's protocol (Bio-Rad 161-0449). The remaining aliquots wereprocessed for LC-MS/MS analysis. Proteins were reduced with 10 mM DTT at56° C. for 1 h, alkylated with 22.5 mM IAA for 30 min at roomtemperature in the dark, and digested with 2.5 μg of trypsin (Promega)at 37° C. overnight. Peptides were desalted using POROS10R2 (AppliedBiosystems) and reconstituted with 0.1% TFA. Peptides were then analyzedby LC-MS/MS on an Orbitrap-XL mass spectrometer (Thermo Scientific), asdescribed in Ficarro et al., Anal. Chem. 81: 3440-3447 (2009). MS/MSspectra were searched against a forward-reversed human NCBI Refseqdatabase using Mascot (Matrix Science, version 2.2.1), and were filteredto a 1% false-discovery rate. Five different criteria were used toselect proteins from MS raw data: 1) molecular weights had to be <28kDa; 2) more than two unique target peptides had to be identified foreach protein; 3) peptides giving an overly weak signal were discarded;4) the protein was cytoplasmic or secreted; 5) nucleoprotein orcytoskeleton proteins were excluded.

Statistical Analysis

Statistical differences between groups were estimated by means of theunpaired Student's t-test, with p≤0.05 being considered significant.Analysis of tumor burden was done using factorial analysis in SPSS 13.0.mRNA expression of CD147 (GSE6477) was measured in BM plasma cells froma normal subject or a MM patient. All experiments were done blindly,without the investigator's knowing the identity of the samples, whichwere labelled only with code numbers.

Example 2 The BCL9 Oncogene Promotes Proliferation ofBone-Marrow-Derived Endothelial Cells

BM angiogenesis is a hallmark of MM progression and a positive correlateof disease activity (FIG. 1A), suggesting that BMECs promote MMprogression. However, the mechanism(s) by which BMECs exert this effectare not fully understood. The BCL9 oncogene is an essentialtranscriptional co-activator of the Wnt/β-catenin complex, and playscritical roles in the pathogenesis of a broad range of human cancers,including colorectal cancer (CC) and MM. Stabilized Alpha-Helix peptidesof BCL9 (SAH-BCL9) that dissociate native β-catenin/BCL9 complexesselectively suppress Wnt transcription, elicit mechanism-basedanti-tumor responses in vitro, and ablate intra-tumoral blood vesselformation in mouse xenograft models of CC and MM 23. These resultssuggested that BCL9 plays a role in MM-associated angiogenesis anddisease progression, prompting us to evaluate its expression in BMECs byimmunohistochemistry (FIGS. 1B and 9A). High levels of BCL9 expressionwere detected in the nucleus of spindled cells in close physical contactwith MM cells (FIG. 1B, left). High expression was observed in all BMbiopsies examined, from normal individuals (NBM) (n=20) (FIG. 1B, right,top-left and FIG. 9A, top) as well as MM patients (n=60) (FIG. 1B,right, top-right and FIG. 9A, bottom). No major differences in BCL9expression in BMECs were noted between normal individuals and MMpatients. Specific expression of BCL9 in BMECs was confirmed by doubleimmunostains, which detected BCL9 and the endothelial cell marker CD34on the same cells (FIG. 1B, right-bottom). Co-expression and nuclearco-localization of BCL9 and β-catenin in two primary BMECs isolated fromMM patients (PBMEC #1 and PBMEC #2), as well as in two established celllines BMEC-60 and BMEC-1, was confirmed by immunoblotting (FIG. 1C) anddouble immunofluorescence (FIG. 1D) analysis. Knockdown of BCL9 inBMEC-60, BMEC-1 and PBMEC #1 cells using previously validated shRNAlentiviral approaches (BCL9-shRNAs) (FIG. 9B) 19 was associated with asignificant decrease in Wnt reporter activity (FIG. 1E) and cellproliferation (FIG. 9C). Consistent with our previous in vivo studies,proliferation of BMECs in culture was likewise inhibited by SAH-BCL9(FIG. 1F).

Example 3 Bone Marrow Endothelial Cells (BMECs) Promote Proliferationand Survival of MM Cells

For a long time, BM-derived stromal cells were considered to be the mainand only cell type with which MM cells interact functionally. However,once BM angiogenesis was recognized as a positive correlate of diseaseactivity (FIG. 1A), it became clear that BMECs contribute to MMprogression. To understand the molecular mechanisms by which BMECspromote MM progression and to evaluate the possible role of BCL9 in thisprocess, we performed biochemical and functional assays of co-culturedcells. As determined by immunoblot analysis of total protein extracts,incubation of MM cells in the presence of BMEC-60 cells activatesseveral signaling pathways compared with MM cells incubated alone (FIG.2A). Among these pathways were found to be the Wnt/β-catenin, STAT3,AKT, and ERK pathways, which are known to promote survival,proliferation, and migration of MM cells. Similar changes were observedwhen MM and BMEC-60 cells were co-cultured in separate chambers (i.e.transwell assay) (FIG. 2B), indicating that soluble factor(s) secretedby BMEC-60 cells promote(s) these signaling changes. Primary BMECs wereas effective as BMEC-60 cells in secreting this factor(s) and promotingsignaling changes (FIG. 2C). In addition, co-culture with BMEC-60 cellslikewise promoted proliferation of MM cells (FIG. 2D) and MM primarytumors (FIG. 2E), and elicited drug resistance in MM1S cells (FIG. 2F).Primary BMECs were as effective as BMEC-60 cells in promotingproliferation of H929 and MM1S cells (FIG. 2G). Furthermore, knockdownof BCL9 in BMEC-60 cells was associated with decreased pSTAT3, pAKT, andpERK activation in transwell assays (FIG. 2H) using H929 and MM cells(FIG. 2H), and was associated with lower proliferation of co-cultured MMcells (FIG. 1).

Example 4 Bone Marrow Endothelial Cells Promote Migration and BoneMarrow Colonoization of Multiple Myeloma Cells

Since activation of the ERK pathway has been previously implicated inpromoting cell migration, our finding of enhanced pERK expression in MMcells co-cultured with BMEC-60 cells (FIGS. 2A, B, H) prompted us to askwhether this activation was also associated with increased migration. Invitro transwell assays revealed that conditioned medium (CM) derivedfrom cultures of BMEC-60 cells (FIG. 3A) or primary BMECs (FIG. 3B), butnot from BM derived stromal HS5 cells (FIG. 3C), promoted migration ofMM1S cells labeled with luciferase (MM1S-luc), compared with the samecells incubated without CM (FIG. 3A). Protease treatment (i.e.,Proteinase K) of BMEC-60-derived CM (FIG. 3A) markedly reduced migrationof MM1S-luc-cells. Migration of other representative MM cell lines(i.e., U266 and RPMI) was also significantly enhanced by CM derived fromcultures from primary BMECs, but not primary BMSCs, isolated from thesame patient or from HS5 cells (FIGS. 9D, E). Furthermore, migration ofMM1S-luc cells was enhanced to a greater degree by primary BMECs than byprimary BMSCs isolated from the same MM patient (FIG. 3C).

To further investigate the role of BMECs in the migration andchemoattraction of MM cells in vivo, we used a scaffold mouse xenograftsystem. Fibronectin-precoated scaffolds were cultured alone or in thepresence of BMEC-60 or HS5 cells for two weeks until uniform coating ofthe scaffold meshwork was observed. Scaffolds were implantedsubcutaneously at different sites in the flank of the samenon-γ-irradiated CB17.Cg-PrkdcscidLystbg-J/Crl mouse, as depicted inFIG. 3D. Two weeks after implantation when growth of host connectivetissue and blood vessels surrounding the scaffolds was demonstrable (asdetermined in pilot experiments), 2×10⁶ MM1S-luc cells were injectedinto the tail vein. In vivo tumor growth within each scaffold wasfollowed over time by xenogen whole-body imaging. Since transplantedhuman MM cell lines can grow within the BM of γ-irradiated mice,non-irradiated mice were used to reduce the background signal and delaythe spread of MM1S-luc cells to the spine. In three independentexperiments, we consistently observed that only scaffolds coated withBMEC-60 cells could support growth of MM1S-luc cells, whereas MM1S-luccells failed to propagate either in scaffolds without cells or in thosecoated with HS5 cells (FIGS. 3E-G). Four weeks post-transplantation,scaffolds were removed and subjected to histological (FIG. 3G, top) andimmunohistochemical (FIG. 3G, bottom) analysis, confirming the presenceof HS5 or BMEC-60 cells within the scaffolds and the infiltration ofMM1S-luc cells in scaffolds coated with BMEC-60 cells, but not in thosewithout cells. Only rare scattered MM1S-luc cells were observed inscaffolds coated with HS5 cells (see also FIG. 9F for triplicateexperiments). Migration and growth of H929-luc cells were similarlyobserved in scaffolds coated with primary PBMECs, but not in uncoatedscaffolds (FIGS. 9G-I). Furthermore, migration and growth of primary MMcells in scaffolds coated with BMEC-60 cells were observed (FIG. 10A),albeit at much lower frequency than that of MM1S-luc cells (2/10 vs.2/2). Since MM primary cells are not susceptible to transduction withlentivirally expressed luciferase, growth of these cells in the scaffoldwas evaluated only by histologic examination of scaffolds after fourweeks of tail-vein injection (FIG. 10A). Primary BMECs were observed tobe much more efficient than primary BMSCs from the same patient's BM inpromoting migration and growth of MM1S-luc cells within scaffolds (FIGS.3H-J).

Example 5 BCL9 Knockdown in BMECs Reduces Migration and Growth of MMCells

We knocked down expression of BCL9 in BMECs (FIG. 9B) to investigatewhether BCL9 was needed to promote the migration and growth of MM cells.In vitro migration of MM1S-luc (FIG. 4A) and H929 cells (FIG. 10B) wassignificantly reduced when co-cultured with BMEC-60 or BMEC-1 cellslentivirally transduced with BCL9-shRNAs. In vivo migration and growthof MM1S-luc cells within scaffolds were also inhibited when BMEC-60cells were lentivirally transduced with BCL9-shRNA. Migration and growthwere similarly observed within control scaffolds coated with BMEC-60cells lentivirally transduced with control shRNAs (FIGS. 4B, C and 10C,D). Histological and immunohistochemical analysis at the end of theexperiment confirmed uniform scaffold coating by bothBMEC-60-control-shRNA (FIG. 4D top) and BMEC-60-BCL9-shRNA (FIG. 4D,bottom) cells, as well as the absence of MM1S-luc cells in scaffoldscoated with BMEC-60-BCL9-shRNA cells, ruling out the possibility thatthe decrease in growth of MM cells within the scaffolds when coated withBMEC-60 cells transduced with BCL9-shRNA is due to a proportionallydecrease in number of cells as a consequence of a decrease inendothelial cell proliferation (FIG. 2I). Similar results were observedwith luciferase-labeled H929 cells using scaffolds coated with BMEC-60cells (FIGS. 10E-G).

Example 6 Proteomic Analysis Identifies eCyPA and eCyPB as SignalingFactors Secreted by BMECs

The foregoing results prompted us to perform proteomic analysis toidentify signaling molecules secreted by BMECs, whose expression isregulated by BCL9, and that promote chemoattraction, migration, andproliferation of MM cells. Silver-stained agarose gels revealedqualitative and quantitative differences, particularly amonglower-molecular weight proteins, in CM from BMEC-60 cells lentivirallytransduced with control-shRNAs or BCL9-shRNAs, as compared with mediumalone. The same was observed in HS5 cells, indicating secretion of adiscrete protein by the control BMEC-60 cells, but not the others (FIG.10H). The absence of lower-molecular weight bands after 1 hr ofincubation (data not shown) in the presence or absence of 10% fetalbovine serum (FBS) rules out the possibility that these differences arerelated to FBS. To identify the low-molecular weight proteins (<28 kD)secreted by BMEC-60 cells, we performed proteomic analysis of the sixmajor bands (bands 1 to 6) with molecular weights of <28 kD fromsilver-stained gels (FIG. 4E, blue), as well as analysis of whole CMfrom BMEC-60 (FIG. 4E, pink), PBMEC #1 (FIG. 4E, yellow), and PBMEC #2(FIG. 4E, green) cells.

Both procedures provided a large number of potential candidates withmolecular weights of <28 kD that are known to be secreted into media,have known signaling functions, and were detected by both procedures inBMEC-60 as well as in primary BMECs. Among these, the most likelycandiates in all cell supernatants were eCyPA and eCyPB (FIG. 4E,intersection and Tables 2 and 3 below). ELISA assays confirmed thepresence of eCyPA and eCyPB in CM from BMEC-60-control-shRNA cells, andshowed that eCyPA and eCyPB secretion were markedly decreased inBEMC-60-BCL9-shRNA and almost absent in CM from HS5 cells (FIG. 4F).ELISA asays also showed that primary BMECs secrete much more eCyPA thanprimary BMSCs isolated from the same MM patient (FIG. 4G). In addition,BMEC-60 cells and primary BMECs secreted much more eCyPA than eCyPB whencompared with HS5 and primary BMSCs (FIGS. 4F, 4G). Immunoblot analysisof several batches of lentivirally transduced BMEC-60 cells confirmedstable BCL9 knockdown and concomitantly decreased cellular CyPA and CyPBexpression in BMEC-60-BCL9-shRNA cells relative to controls (FIG. 10H),indicating that CyPA and CyPB, which are members of the same family ofproteins, have overlapping signaling functions 33 and are bothtranscriptional targets of BCL9. Only cells with >70% knockdown of BCL9expression were selected for in vitro and in vitro assays. Cellular CyPAand CyPB expression was also examined by immunoblot analysis in otherpurified BMECs including BMEC-1 cells, primary BMECs, and primary BMSCsfrom the same MM patient (FIGS. 1C and 9D), and by immunohistochemicalanalysis of BM biopsies from healthy subjects (FIGS. 4H, left and 11A).These experiments confirmed that most BMECs express significant levelsof CyPA, and that CyPB expression is decreased compared with CyPA.

TABLE 2 Proteomics Analysis of BMEC-60 Gene Symbol Description MWProteomics Analysis of Silver Stained Gel Bands Band 1 YWHAB Tyrosine3-Monooxygenase/14-3-3 28082 Da protein beta/alpha YWHAG Tyrosine3-Monooxygenase/14-3-3 28303 Da protien gamma PSMA3 Proteasome subnitalpha type-3 28433 Da Band 2 TAGLN2 Transgelin 2 22391 Da PRDX1Peroxiredoxin 1 22110 Da LZIC Leucine Zipper And CTNNBIP 1 21495 DaDomain Containing Band 3 CyPB Peptidylprolyl Isomerase B 23743 Dapercursor (PPIB) PRDX1 Peroxiredoxin 1 22110 Da TAGLN2 Transgelin 222391 Da Band 4 TANGLN2 Transgelin 2 22391 Da CFL1 Cofilin 1(non-muscle)18502 Da GGCT Gamma-glutamylcyclotransferase 21008 Da Band 5 STMN1Stathmin 1 17303 Da CyPA Peptidylprolyl isomerase A 18012 Da (PPIA) SOD1Superoxide dismutase 1, 15936 Da soluble Band 6 STMN1 Stathmin 1 17303Da ACP1 Acid Phosphatase 1, Soluble 18042 Da SOD1 Superoxide dismutase1, 15936 Da soluble Whole Proteomics Analysis APCS Serum amyloid Pcomponent 25387 Da precursor PRDX6 Peroxiredoxin 6 25035 Da UCHL1Ubiquitin carboxyl-terminal 24824 Da esterase L1 CyPB Peptidylprolylisomerase B 23743 Da precursor (PPIB) GSTP1 Glutathione transferase23356 Da ARHGDIA Rho GDP dissociation inhibitor 23207 Da (GDI) alphaARHGDIB Rho GDP dissociation inhibitor 22988 Da (GDI) beta TAGLN2Transgelin 2 22391 Da PEBP1 Prostatic binding protein 21057 Da GLO1Glyoxalase I 20778 Da HN1L Hemalological and neurological 20063 Daexpressed 1-like CFL1 Cofilin 1 (non-muscle) 18502 Da CyPAPeptidylprolyl isomerase A (PPIA) 18012 Da COTL1 Coactosin-like 1 15945Da SOD1 Superoxide dismutase 1, soluble 15936 Da FABP5 Fatty acidbinding protein 5 15164 Da (psoriasle-associated) PFN1 Profilin 1 15054Da LGALS1 Galectin-1 14716 Da MTPN Myatrophin 12895 Da MIF Marcrophagemigration inhibitory 12476 Da factor TXN Thioreoxin 11737 Da RPLP2Ribosomal protein P2 11665 Da CSTB Cystatin B 11140 Da HSPE1 Heat shock10 kDa protein 1 10932 Da

TABLE 3 Proteomics Analysis of PBMEC #1 and PBMEC #2 Gene SymbolDescription Mw Whole Proteomics Analysis of PBMEC #1 CyPA peptidylprolylisomerase A 18012 Da TXNDC17 thioredoxin-like 5 13941 Da CLIC1 chlorideintracellular channel 1 26923 Da MYL6 myosin, light chain 6, alkali,smooth 16930 Da muscle and non-muscle isoform 1 UCHL1 ubiquitincarboxyl-terminal esterase L1 24824 Da COTL1 coactasin-like 1 15945 DaBASP1 brain abundant, membrane attached signal 22693 Da protein 1 PARK7Parkinson disease protein 7 19891 Da PRDX2 peroxiredoxin 2 isoform a21785 Da GSTM4 glutathione 5-transferase rsu 4 isoform 1 25561 Da GSTP1glutathione transferase 23256 Da LGALS1 galectin-1 14716 Da PRDX1peroxiredoxin 1 22110 Da PEBP1 proslatic binding protein 21057 Da PEA15phosphoprotein enriched in astrocytes 15 15040 Da TPT 1 Tumor protein,translationlly-controlled 1 19595 Da YWHAZ Tyrosine 3/tryptophan5-monooxygenase 27771 Da activation protien, zeta ARHGDIA Rho GDPdissociation inhibitor (GDI) 23207 Da alpha GSTO1Glutathione-S-transferase omega 1 27566 Da PRDX6 Peroxiredoxin 6 25035Da TAGLN Transgelin 22611 Da PGLS 6-phosphogluconolactonase 27547 DaCyPB Peptidylprolyl isomerase B precursor 23743 Da YWHAB Tyrosine3-Monooxygenase/14-3-3 protein 28062 Da beta/alpha YWHAG Tyrosine3-Monooxygenase/14-3-3 protein 28303 Da gamma CAPNS1 Calpain, smallsubunit 1 28316 Da Whole Proteomics Analysis of PBMEC #2 CyPaPeptidylprolyl isomerase A 18012 Da ORM1 Orosomucoid 1 precursor 23512Da BASP1 Brain abundant, membrane attached 22693 Da signal protein 1SDC2 Syndecan 2 precursor 22160 Da APCS Serum amyloid P componentprecursor 25367 Da CALM2 Calmodulin 2 18838 Da CSTB Cystatin B 11140 DaGSTP1 Glutathione transferase 23356 Da LGALS1 Galectin-1 14716 Da PRDX1Peroxiredoxin 1 22110 Da PEBP1 Prostatic binding protein 21057 Da TAGLN2Transgelin 2 22391 Da TTR Transthyrelin precursor 15587 Da YWHAHTyrosine 3-Monooxygenase/14-3-3 protein 26219 Da eta ARHGDIA Rho GPDdissociation inhibitor (GDI) 23407 Da alpha PRDX6 Peroxiredoxin 6 25035Da CyPB Peptidylprolyl isomerase B precursor 25743 Da C1QC Complementcomponent 1, q subcomponent, 25774 Da C chain precursor NME2 Nucleosidediphosphate kinase B 17298 Da PGAM1 Phosphoglycerate mutase 1 26804 Da[Homo sapiens]

Immunohistochemical studies also revealed that BM myeloid cells expresssignificant amounts of CyPA, but not CyPB (FIGS. 4H, 11B); hence,myeloid cells could be an additional source of eCyPA, but not of eCyPB,in the BM niche. In addition, immunohistochemical studies revealed thatalthough MM cells expressed CyPA (FIGS. 4H, right and 11C), theysecreted it at very low levels in comparison with BMECs, or did not doso at all (FIG. 11D). Furthermore, immunohistochemical analysis revealedmuch lower CyPA expression in endothelial cells from other tissuesincluding kidney, liver, and lymph nodes than in BMECs (FIG. 12A).

The foregoing results prompted us to next examine serum levels of eCyPAand eCyPB in MM patients. To this end, serum from BM and PB collectedfrom the same patient at the time of diagnosis were analyzed by ELISA.As shown in FIG. 4I, in MM patients (n=12) eCyPA was significantly moreabundant in BM serum than in serum from PB (BM: 10.54 ng/ml±10.72; PB:1.59 ng/ml±0.94) (P<0.0087). In the same samples, serum levels of eCyPBwere much lower than those of eCyPA (BM: P<0.0032; PB: P<0.0125), and nomajor differences were detected between BM and PB (FIG. 4J). Inaddition, eCyPA levels in PB serum were significantly higher in MM thanMGUS patients (FIG. 12B).

eCyPA levels were also examined in MGUS, SMM, MM patients, and MMtreated patients. FIG. 17 is a graphical representation of an ELISAassay showing eCyPA (ng/ml) in peripheral blood serum from fourdifferent patent groups—MGUS, SMM, MM and MM treated (MGUS: Monoclonalgammopathy of unknown significance; SMM: Smoldering multiple myeloma;MM: multiple myeloma; MM treated: patient under treatment, n=25 pergroup). Serum eCyPA level correlated with progress of multiple myeloma,from MGUS to MM stage, and is decreased after treatment. These resultsshow eCyPA can be used a biomarker to monitor MM progress.

Example 7 eCyPA Promotes Migration, Proliferation and Bone Marrow Homingof Multiple Myeloma Cells

Because of the documented role of CyPA in neutrophil migration, it wasof interest to further probe the role of eCyPA in MM cell migration invitro and in vivo. As shown in FIG. 5A, purified recombinant eCyPApromoted migration of MM1S-luc cells in a time- andconcentration-dependent manner. Although maximal responses were observedat 50 ng/ml, which was used in subsequent assays, significant migrationwas induced at CyPA concentrations at as low as 6 ng/ml, which istypically found in the BM serum of MM patients (FIG. 4I). Theobservation of increased serum levels of eCyPA levels in the BM serum ofMM patients prompted us to examine whether BM serum from MM patientsincreased migration. As shown in FIG. 12C, transwell assays revealedthat BM serum from MM patients promoted migration of MM1S-luc cells toan extent that varied with the concentration of eCyPA. Recombinant CyPAalso induced migration of other MM cell lines (i.e. H929, OPM2, andRPMI) (FIG. 12D) as well as primary MM cells (FIG. 12E). No majordifferences in migration were observed between primary MM cellsincubated in the presence of BMEC-60 cells incubated with 50 ng/ml CyPA(FIG. 12E). Major differences were likewise not observed in themigration of MM1S-luc cells treated with 50 ng/ml CyPA or CyPB,respectively (FIG. 13A). Furthermore, migration of MM1S-luc cells wassignificantly decreased when either BMEC-60 (FIGS. 5B and 13B) or BMEC-1cells (FIGS. 13B, C, D) were transduced with CyPA-shRNAs. As would beexpected from an on-target effect of eCyPA-shRNAs, addition ofrecombinant eCyPA restored migration of MM cells incubated with BMEC-1transduced with CyPA-sh-RNAs (FIG. 13D). Immunoblot analysis of totalprotein extracts from MM cells incubated with eCyPA revealed activationof several signaling pathways as compared with MM cells incubatedwithout eCyPA. Among these were the Wnt/β-catenin, STAT3, AKT, and ERKpathways (FIG. 5C), all of which are known to promote survival,proliferation, and migration of MM cells. We also observed that thehigher-glycosylated form of CD147 (i.e., the active form that inducesmatrix metalloprotease production and metastasis) was increased inCyPA-treated MM cells (FIG. 5C, arrows). Expression of matrixmetalloprotease 9 (MMP-9) itself increased in cells treated with eCyPA(FIG. 5C). CD147 was expressed at very low levels in MM cells incubatedin the absence of eCyPA, whereas incubation with eCyPA dramaticallyincreased its expression in a concentration-dependent (FIG. 5D, top) andtime-dependent (FIG. 5D, bottom) manner. The signaling changes inducedby eCyPA were reversed when MM cells were incubated in the presence ofBMEC-60 cells with knockdown expression of CyPA (FIG. 5E). Proliferationof H929 and MM1S cells was also enhanced by eCyPA in aconcentration-dependent manner (FIG. 5F).

We next examined the role of eCyPA in vivo using the scaffold xenograftmouse model (FIG. 3D). These studies revealed that in vivo migration andgrowth of MM1S-luc cells within the scaffolds was inhibited when thescaffolds were coated with BMEC-60 cells lentivirally transduced withCyPA-shRNA as compared with control cells lentivirally transduced withcontrol-shRNAs (FIGS. 5G-I). Immunohistochemical (FIG. 5I, top) andhistological (FIG. 5I, bottom) analysis at the end of the experimentconfirmed uniform scaffold coating with both BMEC-60-control-shRNA andBMEC-60-CyPA-shRNA cells. The results were replicated in two otherindependent experiments (FIG. 13E top and bottom), and reproduced withH929-luc cells and scaffolds coated with BMEC-60 cells (FIGS. 13F-H).

Example 8 CyPA Promotes Migration and Growth of MM Through the CD147Receptor

Because recent studies have established CD147 as the principal signalingreceptor mediating extracellular chemotaxis by eCyPA, we evaluated CD147expression in MM cells using flow cytometric, immunohistochemical, andgene expression profiling analysis (Affimetrix). These studies showedthat most myeloma cell lines, including MM1S-luc (FIG. 14A) and primarytumors (FIG. 14B), expressed significant amounts of CD147. At the mRNAlevel, CD147 expression was present in most MM samples, withsignificantly higher levels detected in MM samples than in normal plasmacells (FIG. 14C), indicating that CD147 expression increases withdisease progression.

We then evaluated whether CD147 inactivation on MM cells could block theaction of eCyPA. Knockdown of CD174 expression in MM1S and H929 cellswith CD147-shRNAs (FIG. 14D) reduced migration in transwell assays inthe presence of either BMEC-60 cells or CyPA (FIGS. 6A and 14E). Thesignaling changes induced by CyPA in H929 and MM1S cells (FIG. 5C) werereversed when the cells were first transduced with CD147-shRNAs (FIG.6B). We also examined the role of CD147 in vivo using the scaffoldxenograft mouse model (FIG. 3D). As shown in FIGS. 6C-E, knockdown CD147expression in MM1S-luc cells inhibited migration and growth withinscaffolds coated with BMEC-60 cells.

Example 9 Targeting the eCyPA/CD147 Complex for Multiple Myeloma Therapy

To test the targeting of the eCyPA/CD147 complex as a potential strategyfor clinical MM therapy, we first evaluated whether an anti-CD147 Abknown to inhibit neutrophil migration in a mouse model of inflammation35 also inhibits MM cell migration. Transwell assays revealed thateCyPA-induced MM1S-luc cell migration was markedly reduced if the cellswere pre-treated with the CD147 Ab (FIG. 6F). Decreased migration wasassociated with inhibition of CyPA-induced activation of pERK, pSTAT3,pAKT, and PARP (FIG. 14F). PARP activation by CD147 Ab wasconcentration-dependent (FIG. 14G). Interestingly, CXCR4 Ab migration ofMM cells induced by SDF-113 did not inhibit CyPA-induced migration ofMM1S-luc cells (FIG. 14H). Migration of MM1S-luc cells was also markedlyinhibited by the CD147 Ab when cells were incubated in the presence ofprimary BMECs, but not primary BMSCs, isolated from same MM patient(FIG. 6G). In addition, treatment with a CXCR4 Ab did not inhibitmigration of MM1S-luc cells induced by primary BMECs, but markedlyinhibited migration induced by primary BMSCs (FIG. 6G). These resultsprompted us to evaluate secretion of eCyPA and SDF-1 in primary BMECsand primary BMSCs by ELISA. As shown in FIG. 14I, eCyPA was detected inCM from primary BMECs, but not primary BMSCs, and an inverse correlationwas observed with SDF-1. These results suggested that primary BMECs andprimary BMSCs play different roles in MM migration, most likely becausethey secrete different chemo-attractant factors.

Our observation of decreased pSTAT in MM cells either transduced withCD147-shRNA (FIG. 6B) or treated with CD147 Ab (FIG. 14F) prompted us tonext use the scaffold system to evaluate whether CD147 Ab could betherapeutic for MM in vivo. To this end, scaffolds were pre-coated withBMEC-60 cells for two weeks, and then incubated with MM1S-luc cells forone week. When an evenly distributed coating of cells was obtained, thescaffolds were implanted into the flank of non-γ-irradiatedimmunodeficient mice. One week post-implantation the mice were subjectedto whole-body imaging, and mice with comparable tumor burdens wereselected for Ab therapy (FIG. 7A, Day 0). Mice were injected with 100 μlof a solution containing either 100 ng of isotype Ab (control group,n=4) or anti-CD147 Ab (treated group, n=4) s.c. every other day, in aregion adjacent to the scaffolds, and tumor growth within the scaffoldwas evaluated by Xenogen imaging every five days. On day 15 of treatmentthe mice were euthanized, and the scaffolds weresubjected to histologicand immunohistochemical analysis (FIG. 7B). Quantitative Xenogen imagingrevealed that anti-CD147 Ab treatment had significantly decreasedMM1S-luc growth within the scaffolds compared with isotype Ab-treatedmice (FIG. 15A). In addition, more intense immunostaining for caspasewas observed in MM1S-luc cells within scaffolds of mice treated withCD147 Ab (FIG. 7B) than in mice treated with isotype Ab. Analysis ofimmune-mediated activity revealed that complement-dependent cytotoxicitywas not induced by the CD147 Ab (FIG. 15B). Taken together, thesestudies indicated that inhibition of the eCyPA/CD147 axis inhibitsproliferation and induces apoptosis of MM cells, and thus couldrepresent a novel targeted therapy in MM.

Example 10 Decreased CD147 Expression in Circulating MM Cells

Since our previous results indicated that eCyPA secreted by BMECs is animportant driver of BM homing by MM cells via binding of the CD147receptor expressed on MM cells, we measured CD147 expression in plasmacells from the lymph nodes and BM of healthy subjects, as well as incells in plasma cells from BM and PB of MM patients. Due to the paucityof plasma cells in BM and lymph nodes of normal individuals as well astheir relatively small numbers in the PB of MM patients, immunoblottingstudies were not feasible. In these instances, therefore, CD147expression was determined by double-wavelength immunofluorescenceanalysis of cytospin preparations using CD138 as a plasma cell marker.These studies revealed that all BM plasma cells analyzed in 10/10 MMpatients expressed high CD147 levels (FIGS. 7C, top and 15C, top) while9/10 tested samples of PB plasma cells from the same MM patient did not(FIGS. 7C, bottom and 15C, bottom). In contrast, 9/10 normal plasmacells analyzed from BM (FIGS. 7D, top and 15D, top) and 8/10 cellsanalyzed from lymph nodes of every normal subject (FIGS. 7D, bottom and15D, bottom) expressed CD147 at only low or undetectable levels.

Example 11 CLL and LPL Cells Express CD147 and Migrate in Response toeCyPA

Since CLL and LPL are B-cell malignancies that, like MM, preferentiallycolonize the BM, it was of interest to also examine whether cell linesand primary tumor cells derived from patients express CD147, and whethereCyPA is chemotactic to these cells. Immunostains (FIGS. 16A, B, top),and flow cytometry (FIGS. 16A, B, bottom) revealed that most CLL and LPLprimary cells expressed CD147. In addition, the CLL cell line HG3 andprimary tumor CLLPT #1 (FIG. 16C) as well as the LPL cell line BCWM.1and LPLPT #1 (FIG. 16D) used in subsequent in vitro migration studiesexpressed high levels of CD147. Furthermore, both BMEC-60-derived CM andeCyPA enhanced migration of CLL (FIG. 16E, bottom) and LPL (FIG. 16F,bottom) cells in transwell assays.

On the basis of the present findings and the generally recognizedtrafficking model of normal lymphocytes and BM recruitment ofhematopoietic stem cells (HSCs), we propose a mechanism that couldexplain both the preferential BM colonization of MM cells during earlystages of disease and loss of preferential homing during the late stagesof disease progression (e.g., plasma cell leukemia) (FIG. 8). In thismodel, normal plasma cells do not accumulate in the BM because theydon't express CD147 39 (FIG. 7D), whereas MM cells can accumulate in BMbecause they do express CD147 39 (FIG. 7C), the known receptor for eCyPA33 which serves as a chemoattractant promoting migration of MM cellsalong a concentration gradient between the PB and BM (FIG. 4I). Thisgradient is generated because endothelial cells from the BM, but notthose from other organ sites, produce and secrete eCyPA in significantamounts. In the BM, the effect of eCyPA from endothelial cells can beenhanced by local production and secretion by myeloid cells (FIG. 11B).Although MM cells also express CyPA (FIG. 4H), they secrete it at verylow rates relative to BMECs (FIG. 11D), indicating that MM are lessefficient than BMECs in terms of Rho-kinase-mediated CyPA secretion, andthat MM-derived eCyPA does not play a significant role in MM recruitmentto the BM, at least during initial stages of disease, when tumor burdenis low. As MM cells circulate in the PB and encounter progressivelyhigher concentrations of eCyPA, binding of eCyPA to CD147 furtherincreases CD147 expression by MM cells (FIG. 5D), thereby enhancingtheir migration. Additionally, extravasation of MM cells, required forpenetration through the sub-endothelial basement membrane, is mediatedby the production of proteolytic enzymes such as MMP-9, whose level isknown to rise when eCyPA binds to CD147 (FIG. 5C). Once MM cells arewithin the BM niche, colonization and growth is further enhanced bydirect exocrine action of eCyPA and by local production and secretion ofother growth factors and chemoattractants (e.g. laminin-1, MIP-1α,SDF-1, MCP-1, VEGF, IGF-1). Therefore, selective colonization of MMcells induced by eCyPA is the consequence of selective BM homing (i.e.,selective migration of MM cells through the blood to the BM niche),followed by selective survival and growth within the BM niche. It isalso possible that eCyPA in the BM serves as a “retention signal” for MMcells, which needs to be further investigated. During MM progression,the number of circulating MM cells may increase because of genetic andepigenetic changes in the tumor that decrease CD147 expression (FIG.7C). As a consequence MM cells, now unable to respond to eCyPA, canleave the BM to colonize extramedullary sites.

The role we have uncovered for the eCyPA/CD147 complex in MM identifiesthis interaction as an attractive target for therapeutic intervention.Agents potentially disruptive of this interaction can be grouped intothose that can directly target CD147, eCyAP, or their mutualinteraction, or those which act indirectly via inhibition of theWnt/β-catenin/BCL9 transcriptional complex. Indeed, using CD147 Ab as atool to block eCyPA- and BMECs-induced migration of MM cells (FIG. 6G),we provide here the first evidence that inhibition of the eCyPA/CD147complex is associated with increased caspase 3 activity (FIG. 7B) andanti-MM activity (FIG. 15A). Such agents may also be useful for thetreatment of other B-cell malignancies, such as CLL and LPL, whichsimilarly express CD147 and respond to eCyPA (FIG. 16). Interestingly,CD147 inhibition using an anti-CD147 monoclonal antibody has beenreported to afford significant symptom relief in murine models of acutelung inflammation, asthma and rheumatoid arthritis, and hepatocellularcarcinoma.

In summary, our studies have uncovered a pivotal role of the eCyPA/CD147axis in the functional interaction between MM cells and BMECs thatcontributes to BM homing as well as initiation and/or progression of MM,and have established an important functional link between the oncogenicWnt/β-catenin/BCL9 pathway and the eCyPA/CD147 system which implicateschronic inflammation in the pathogenesis of MM. Translationalapplications of our studies to the clinical setting include: (1)identifying biomarkers of disease progression within the eCyPA/CD147cascade, and (2) developing novel therapies which target eCyPA/CD147interactions in MM and other B-cell malignancies that preferentiallycolonize and grow within the BM niche.

It is to be understood that the foregoing description is intended toillustrate and not to limit the scope of the invention, which is definedby the scope of the appended claims. Other embodiments are within thescope of the following claims.

What is claimed is:
 1. A method for identifying a therapeutic agent fortreating multiple myeloma (MM), the method comprising: (a) providing afirst polypeptide comprising a CD147 polypeptide and a secondpolypeptide comprising an extracellular cyclophilin A (eCyPA)polypeptide sequence under conditions that allow for binding of theCD147 polypeptide and the eCyPA sequence; (b) contacting the complexwith a test agent; (c) determining whether the test agent disruptsbinding of the first and second polypeptide, wherein disruption of thebinding of the first polypeptide and second polypeptide by the testagent indicates the test agent is a putative therapeutic agent fortreating MM; (d) contacting the putative therapeutic agent with amultiple myeloma (MM) cell population expressing CD147; and (e)determining whether the putative therapeutic agent inhibits migration ofMM cells from the MM cell population into bone marrow, whereininhibition of MM cell migration indicates the test agent is atherapeutic agent for treating MM.
 2. The method of claim 1, wherein theCD147 polypeptide is provided in a cell expressing a CD147 polypeptidesequence.
 3. The method of claim 2, wherein the cell expressing a CD147polypeptide sequence is provided in vitro.
 4. The method of claim 2,wherein the cell expressing a CD147 polypeptide sequence is provided invivo.
 5. The method of claim 2, wherein the cell expressing a CD147polypeptide sequence is a human cell.
 6. The method of claim 2, whereinthe cell expressing a CD147 polypeptide sequence is a lymphocyte,optionally wherein the lymphocyte is a B cell.
 7. The method of claim 2,wherein the cell expressing a CD147 polypeptide sequence is a multiplemyeloma cell.
 8. The method of claim 1, wherein the amino acid sequenceof the eCyPA polypeptide is set forth in SEQ ID NO:1.
 9. The method ofclaim 1, wherein the amino acid sequence of the CD147 polypeptide is setforth in SEQ ID NO:2.
 10. The method of claim 1, wherein the test agentis a nucleic acid, a polypeptide, a small molecule, or combinationsthereof.
 11. The method of claim 10, wherein the test agent is a nucleicacid, and wherein the nucleic acid is an inhibitory nucleic acid. 12.The method of claim 11, wherein the inhibitory nucleic acid is a triplexforming oligonucleotide, an aptamer, a ribozyme, an antisense RNA, ashort interfering RNA (siRNA), or a micro-RNA (miRNA).
 13. The method ofclaim 10, wherein the test agent is a polypeptide, and wherein thepolypeptide is an antibody or an antigen-binding derivative thereof. 14.The method of claim 13, wherein antibody or an antigen-bindingderivative thereof specifically binds to CyPA.
 15. The method of claim14, wherein the antibody or antigen-binding derivative thereof ishumanized.
 16. The method of claim 1, wherein disruption of the bindingof the first polypeptide and second polypeptide by the test agentindicates the test agent is a putative therapeutic agent for treatingchronic lymphocytic leukemia (CLL) or lymphoplasmacytic lymphoma (LPL).